Novel low density lipoprotein binding proteins and their use in diagnosing and treating atherosclerosis

ABSTRACT

Isolated polynucleotides encoding novel polypeptides which are capable of binding to native and methylated LDL (low density lipoprotein), the isolated polypeptides, called LBPs (LDL binding proteins), and biologically active fragments and analogs thereof, are described. Also described are methods for determining if an animal is at risk for atherosclerosis, methods for evaluating an agent for use in treating atherosclerosis, methods for treating atherosclerosis, and methods for treating a cell having an abnormality in structure or metabolism of LBP. Pharmaceutical compositions and vaccine compositions are also provided.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/031,930 filed Nov. 27, 1996, and U.S. ProvisionalApplication No. 60/048,547 filed Jun. 3, 1997.

FIELD OF THE INVENTION

[0002] This invention relates to novel polypeptides (LBPs) which bind tolow density lipoprotein (LDL), polynucleotides which encode thesepolypeptides, and treatments, diagnoses and therapeutic agents foratherosclerosis.

BACKGROUND OF THE INVENTION

[0003] Atherosclerosis is the principal cause of heart attacks andstrokes. It has been reported that about 50% of all deaths in the UnitedStates, Europe and Japan are due to atherosclerosis. Atheroscleroticlesions in the arterial wall characterize atherosclerosis. Cholesterylesters (CE) are present in these atherosclerotic lesions. Low densitylipoprotein (LDL) has been shown to be the major carrier of plasma CE,and has been implicated as the agent by which CE enter theatherosclerotic lesions.

[0004] Scattered groups of lipid-filled macrophages, called foam cells,are the first visible signs of atherosclerosis and are described as typeI lesions. These macrophages are reported to contain CE derived fromLDL. The macrophages recognize oxidized LDL, but not native LDL, andbecome foam cells by phagocytosing oxidized LDL. Larger, more organizedcollections of foam cells, fatty streaks, represent type II lesions.These lesions further develop into complex lesions called plaques, whichcan result in impeding the flow of blood in the artery.

[0005] It is widely believed that accumulation of LDL in the arterydepends on the presence of functionally modified endothelial cells inthe arterial wall. It has been reported in animal models ofatherosclerosis that LDL, both native LDL and methylated LDL,accumulates focally and irreversibly only at the edges of regeneratingendothelial islands in aortic lesions, where functionally modifiedendothelial cells are present, but not in the centers of these islandswhere endothelial regeneration is completed. Similarly, LDL accumulatesin human atherosclerotic lesions. The mechanism by which the LDLaccumulates focally and irreversibly in arterial lesions has notheretofore been understood.

SUMMARY OF THE INVENTION

[0006] It is an object of the invention to provide polypeptides whichbind to LDL.

[0007] It is yet another object of the invention to provide a method fordetermining if an animal is at risk for atherosclerosis.

[0008] It is yet another object of the invention to provide a method forevaluating an agent for use in treating atherosclerosis.

[0009] It is yet another object of the invention to provide a method fortreating atherosclerosis.

[0010] Still another object of the invention is to utilize an LBP (lowdensity lipoprotein binding protein) gene and/or polypeptide, orfragments, analogs and variants thereof, to aid in the treatment,diagnosis and/or identification of therapeutic agents foratherosclerosis.

[0011] In one aspect, the invention features an isolated polynucleotidecomprising a polynucleotide encoding the polypeptide comprising theamino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 orSEQ ID NO:9; or a polynucleotide capable of hybridizing to and which isat least about 95% identical to any of the above polynucleotides andwherein the encoded polypeptide is capable of binding to LDL; or abiologically active fragment of any of the above polynucleotides whereinthe encoded polypeptide is capable of binding to LDL.

[0012] In certain embodiments, the polynucleotide comprises the nucleicacid sequence as set forth in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 orSEQ ID NO:18.

[0013] Another aspect of the invention is an isolated polypeptidecomprising a polypeptide having the amino acid sequence as set forth inSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9; or a polypeptide which isat least about 95% identical to any of the above polypeptides andwherein the polypeptide is capable of binding to LDL; or a biologicallyactive fragment of any of the above polypeptides wherein the fragment iscapable of binding to LDL.

[0014] Another aspect of the invention is a method for determining if ananimal is at risk for atherosclerosis. An animal is provided. An aspectof LBP metabolism or structure is evaluated in the animal. Anabnormality in the aspect of LBP metabolism or structure is diagnosticof being at risk for atherosclerosis.

[0015] Another aspect of the invention is a method for evaluating anagent for use in treating atherosclerosis. A test cell, cell-free systemor animal is provided. An agent is provided. The agent is administeredto the test cell, cell-free system or animal in a therapeuticallyeffective amount. The effect of the agent on an aspect of LBP metabolismor structure is evaluated. A change in the aspect of LBP metabolism orstructure is indicative of the usefulness of the agent in treatingatherosclerosis.

[0016] Another aspect of the invention is a method for evaluating anagent for the ability to alter the binding of LBP polypeptide to abinding molecule, e.g., native LDL, modified LDL, e.g., methylated LDLor oxidized LDL, or an arterial extracellular matrix structuralcomponent. An agent is provided. An LBP polypeptide is provided. Abinding molecule is provided. The agent, LBP polypeptide and bindingmolecule are combined. The formation of a complex comprising the LBPpolypeptide and binding molecule is detected. An alteration in theformation of the complex in the presence of the agent as compared to inthe absence of the agent is indicative of the agent altering the bindingof the LBP polypeptide to the binding molecule.

[0017] Another aspect of the invention is a method for evaluating anagent for the ability to bind to an LBP polypeptide. An agent isprovided. An LBP polypeptide is provided. The agent is contacted withthe LBP polypeptide. The ability of the agent to bind to the LBPpolypeptide is evaluated.

[0018] Another aspect of the invention is a method for evaluating anagent for the ability to bind to a nucleic acid encoding an LBPregulatory sequence. An agent is provided. A nucleic acid encoding anLBP regulatory sequence is provided. The agent is contacted with thenucleic acid. The ability of the agent to bind to the nucleic acid isevaluated.

[0019] Another aspect of the invention is a method for treatingatherosclerosis in an animal. An animal in need of treatment foratherosclerosis is provided. An agent capable of altering an aspect ofLBP structure or metabolism is provided. The agent is administered tothe animal in a therapeutically effective amount such that treatment ofthe atherosclerosis occurs. In certain embodiments, the agent is an LBPpolypeptide, e.g., LBP-1, LBP-2 or LBP-3, or a biologically activefragment or analog thereof. In certain embodiments, the agent is apolypeptide of no more than about 100, 50, 30, 20, 10, 5, 4, 3 or 2amino acid residues in length. In certain embodiments, the agent is apolypeptide having an amino acid sequence that includes at least about20%, 40%, 60%, 80%, 90%, 95% or 98% acidic amino acid residues.

[0020] Another aspect of the invention is a method for treating ananimal at risk for atherosclerosis. An animal at risk foratherosclerosis is provided. An agent capable of altering an aspect ofLBP structure or metabolism is provided. The agent is administered tothe animal in a therapeutically effective amount such that treatment ofthe animal occurs.

[0021] Another aspect of the invention is a method for treating a cellhaving an abnormality in structure or metabolism of LBP. A cell havingan abnormality in structure or metabolism of LBP is provided. An agentcapable of altering an aspect of LBP structure or metabolism isprovided. The agent is administered to the cell in a therapeuticallyeffective amount such that treatment of the cell occurs.

[0022] Another aspect of the invention is a pharmaceutical compositionfor treating atherosclerosis in an animal comprising a therapeuticallyeffective amount of an agent, the agent being capable of altering anaspect of LBP metabolism or structure in the animal so as to result intreatment of the atherosclerosis, and a pharmaceutically acceptablecarrier.

[0023] Another aspect of the invention is a vaccine composition fortreating atherosclerosis in an animal comprising a therapeuticallyeffective amount of an agent, the agent being capable of altering anaspect of LBP metabolism or structure in the animal so as to result intreatment of the atherosclerosis, and a pharmaceutically acceptablecarrier.

[0024] Another aspect of the invention is a method for diagnosingatherosclerotic lesions in an animal. An animal is provided. A labeledagent capable of binding to LBP, e.g., LBP-1, LBP-2 or LBP-3, present inatherosclerotic lesions is provided. The labeled agent is administeredto the animal under conditions which allow the labeled agent to interactwith the LBP so as to result in labeled LBP. The localization orquantification of the labeled LBP is determined by imaging so as todiagnose the presence of atherosclerotic lesions in the animal.

[0025] Another aspect of the invention is a method for immunizing ananimal against an LBP, e.g., LBP-1, LBP-2 or LBP-3, or fragment oranalog thereof. An animal having LDL is provided. The LBP or fragment oranalog thereof is administered to the animal so as to stimulate antibodyproduction by the animal to the LBP or fragment or analog thereof suchthat binding of the LBP to the LDL is altered, e.g., decreased orincreased.

[0026] Another aspect of the invention is a method of making a fragmentor analog of LBP polypeptide, the fragment or analog having the abilityto bind to native LDL and to modified LDL, e.g., methylated LDL,oxidized LDL, acetylated LDL, or cyclohexanedione-treated LDL. An LBPpolypeptide is provided. The sequence of the LBP polypeptide is altered.The altered LBP polypeptide is tested for the ability to bind tomodified LDL and native LDL.

[0027] Yet another aspect of the invention is a method for isolating acDNA encoding an LBP. A cDNA library is provided. The cDNA library isscreened for a cDNA encoding a polypeptide which binds to native LDL andmodified LDL, e.g., methylated LDL or oxidized LDL. The cDNA whichencodes the polypeptide is isolated, the cDNA encoding an LBP.

[0028] The above and other features, objects and advantages of thepresent invention will be better understood by a reading of thefollowing specification in conjunction with the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIG. 1 depicts the amino acid sequence of rabbit LBP-1 (SEQ IDNO:1). Differences in amino acids between rabbit and human LBP-1 aredepicted in bold type.

[0030]FIG. 2 depicts the amino acid sequence of rabbit LBP-2 (SEQ IDNO:2). Differences in amino acids between rabbit and human LBP-2 aredepicted in bold type.

[0031]FIG. 3 depicts the amino acid sequence of amino acids 86 to 317 ofrabbit LBP-2 (SEQ ID NO:3).

[0032]FIG. 4 depicts the amino acid sequence of amino acids 66 to 317 ofrabbit LBP-2 (SEQ ID NO:4).

[0033]FIG. 5 depicts the amino acid sequence of rabbit LBP-3 (SEQ IDNO:5). Differences in amino acids between rabbit and human LBP-3 aredepicted in bold type.

[0034]FIG. 6 depicts the amino acid sequence of human LBP-1 (SEQ IDNO:6). Differences in amino acids between rabbit and human LBP-1 aredepicted in bold type.

[0035]FIG. 7 depicts the amino acid sequence of human LBP-2 (SEQ IDNO:7). Differences in amino acids between rabbit and human LBP-2 aredepicted in bold type.

[0036]FIG. 8 depicts the amino acid sequence of human LBP-3 (SEQ IDNO:8). Differences in amino acids between rabbit and human LBP-3 aredepicted in bold type.

[0037]FIG. 9 depicts the amino acid sequence of amino acids 14 to 33 ofhuman or rabbit LBP-1, called EHF-1 (SEQ ID NO:9).

[0038]FIG. 10 depicts the cDNA sequence encoding rabbit LBP-1 (SEQ IDNO:10) and the corresponding amino acid sequence. Differences in aminoacids between rabbit and human LBP-1 are depicted in bold type.

[0039]FIG. 11 depicts the cDNA sequence encoding rabbit LBP-2 (SEQ IDNO:11) and the corresponding amino acid sequence. Differences in aminoacids between rabbit and human LBP-2 are depicted in bold type.

[0040]FIG. 12 depicts the cDNA sequence 256 to 1617 of rabbit LBP-2 (SEQID NO:12) and the corresponding amino acid sequence.

[0041]FIG. 13 depicts the cDNA sequence 196 to 1617 of rabbit LBP-2 (SEQID NO:13) and the corresponding amino acid sequence.

[0042]FIG. 14 depicts the cDNA sequence encoding rabbit LBP-3 (SEQ IDNO:14) and the corresponding amino acid sequence. Differences in aminoacids between rabbit and human LBP-3 are depicted in bold type.

[0043]FIG. 15 depicts the cDNA sequence encoding human LBP-1 (SEQ IDNO:15) and the corresponding amino acid sequence. Differences in aminoacids between rabbit and human LBP-1 are depicted in bold type.

[0044]FIG. 16 depicts the cDNA sequence encoding human LBP-2 (SEQ IDNO:16) and the corresponding amino acid sequence. Differences in aminoacids between rabbit and human LBP-2 are depicted in bold type.

[0045]FIG. 17 depicts the cDNA sequence encoding human LBP-3 (SEQ IDNO:17) and the corresponding amino acid sequence. Differences in aminoacids between rabbit and human LBP-3 are depicted in bold type.

[0046]FIG. 18 depicts the cDNA sequence encoding BHF-1 (SEQ ID NO:18).

[0047]FIG. 19 corresponds to the amino acid sequence of rabbit LBP-1(top sequence) in alignment with the amino acid sequence of human LBP-1(bottom sequence).

[0048]FIG. 20 corresponds to the amino acid sequence of rabbit LBP-2(top sequence) in alignment with the amino acid sequence of human LBP-2(bottom sequence).

[0049]FIG. 21 corresponds to the amino acid sequence of rabbit LBP-3(top sequence) in alignment with the amino acid sequence of human LBP-3(bottom sequence).

DETAILED DESCRIPTION

[0050] In accordance with aspects of the present invention, there areprovided novel mature human and rabbit polypeptides, LBP-1, LBP-2 andLBP-3, and biologically active analogs and fragments thereof, and thereare provided isolated polynucleotides which encode such polypeptides.LBP is an abbreviation for low density lipoprotein (LDL) bindingprotein. The terms polynucleotide, nucleotide and oligonucleotide areused interchangeably herein, and the terms polypeptides, proteins andpeptides are used interchangeably herein.

[0051] This invention provides for an isolated polynucleotide comprisinga polynucleotide encoding the polypeptide having the amino acid sequenceof rabbit LBP-1 as set forth in FIG. 1 (SEQ ID NO:1); rabbit LBP-2 asset forth in FIG. 2 (SEQ ID NO:2); 86 to 317 of rabbit LBP-2 as setforth in FIG. 3 (SEQ ID NO:3); 66 to 317 of rabbit LBP-2 as set forth inFIG. 4 (SEQ ID NO:4); rabbit LBP-3 as set forth in FIG. 5 (SEQ ID NO:5);human LBP-1 as set forth in FIG. 6 (SEQ ID NO:6); human LBP-2 as setforth in FIG. 7 (SEQ ID NO:7); human LBP-3 as set forth in FIG. 8 (SEQID NO:8); 14 to 33 of human or rabbit LBP-1, called BHF-1, as set forthin FIG. 9 (SEQ ID NO:9); a polynucleotide capable of hybridizing to andwhich is at least about 80% identical, more preferably at least about90% identical, more preferably yet at least about 95% identical, andmost preferably at least about 98% identical to any of the abovepolynucleotides, and wherein the encoded polypeptide is capable ofbinding to LDL; or a biologically active fragment of any of the abovepolynucleotides wherein the encoded polypeptide is capable of binding toLDL.

[0052] This invention also includes an isolated polynucleotidecomprising a polynucleotide encoding the polypeptide having amino acidresidues 8-22 (SEQ ID NO:19), 8-33 (SEQ ID NO:20), 23-33 (SEQ ID NO:21)or 208-217 (SEQ ID NO:22) of human LBP-2 as set forth in FIG. 7 (SEQ IDNO:7); amino acid residues 14-43 (SEQ ID NO:23) or 38-43 (SEQ ID NO:24)of rabbit or human LBP-1 as set forth in FIG. 1 (SEQ ID NO:1) and FIG. 6(SEQ ID NO:6); amino acid residues 105-120 (SEQ ID NO:25), 105-132 (SEQID NO:26), 121-132 (SEQ ID NO:27) or 211-220 (SEQ ID NO:28) of rabbitLBP-2 as set forth in FIG. 2 (SEQ ID NO:2); amino acid residues 96-110(SEQ ID NO:29) of rabbit LBP-3 as set forth in FIG. 5 (SEQ ID NO:5);amino acid residues 53-59 (SEQ ID NO:41) of human LBP-3 as set forth inFIG. 8 (SEQ ID NO:8); a polynucleotide capable of hybridizing to andwhich is at least about 80% identical, more preferably at least about90% identical, more preferably yet at least about 95% identical, andmost preferably at least about 98% identical to any of the abovepolynucleotides, and wherein the encoded polypeptide is capable ofbinding to LDL; or a biologically active fragment of any of the abovepolynucleotides wherein the encoded polypeptide is capable of binding toLDL.

[0053] By a polynucleotide encoding a polypeptide is meant apolynucleotide which includes only coding sequence for the polypeptide,as well as a polynucleotide which includes additional coding and/ornon-coding sequences. Thus, e.g., the polynucleotides which encode forthe mature polypeptides of FIGS. 1-9 (SEQ ID NOS:1-9) may include onlythe coding sequence for the mature polypeptide; the coding sequence forthe mature polypeptide and additional coding sequence such as a leaderor secretory sequence or a proprotein sequence; the coding sequence forthe mature polypeptide (and optionally additional coding sequence) andnon-coding sequence, such as introns or non-coding sequences 5′ and/or3′ of the coding sequence for the mature polypeptide. Thepolynucleotides of the invention are also meant to includepolynucleotides in which the coding sequence for the mature polypeptideis fused in the same reading frame to a polynucleotide sequence whichaids in expression and/or secretion of a polypeptide from a host cell,e.g., a leader sequence. The polynucleotides are also meant to includepolynucleotides in which the coding sequence is fused in frame to amarker sequence which, e.g., allows for purification of the polypeptide.

[0054] The polynucleotides of the present invention may be in the formof RNA, DNA or PNA, e.g., cRNA, cDNA, genomic DNA, or synthetic DNA, RNAor PNA. The DNA may be double-stranded or single stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand.

[0055] In preferred embodiments, the polynucleotide comprises thenucleic acid of rabbit LBP-1 as set forth in FIG. 10 (SEQ ID NO:10);rabbit LBP-2 as set forth in FIG. 11 (SEQ ID NO:11); nucleotide 256 to1617 of rabbit LBP-2 as set forth in FIG. 12 (SEQ ID NO:12); nucleotide196 to 1617 of rabbit LBP-2 as set forth in FIG. 13 (SEQ ID NO:13);rabbit LBP-3 as set forth in FIG. 14 (SEQ ID NO:14); human LBP-1 as setforth in FIG. 15 (SEQ ID NO:15); human LBP-2 as set forth in FIG. 16(SEQ ID NO:16); human LBP-3 as set forth in FIG. 17 (SEQ ID NO:17); ornucleotide 97 to 156 of rabbit LBP-1 or nucleotide 157 to 216 of humanLBP-1, (BHF-1), as set forth in FIG. 18 (SEQ ID NO:18).

[0056] In other preferred embodiments, the polynucleotide comprises thenucleic acid as set forth in SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32,SEQ ID NO:33 SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQID NO:38, SEQ ID NO:39, SEQ ID NO:40 or SEQ ID NO:42.

[0057] The coding sequence which encodes the mature polypeptide may beidentical to the coding sequences shown in FIGS. 10-18 (SEQ IDNOS:10-18) or SEQ ID NOS:30-40 or 42, or may be a different codingsequence which coding sequence, as a result of the redundancy ordegeneracy of the genetic code, encodes the same mature polypeptides asthe DNA of FIGS. 10-18 (SEQ ID NOS:10-18) and SEQ ID NOS: 30-40 and 42.

[0058] This invention also includes recombinant vectors comprising thepolynucleotides described above. The vector can be, e.g., a plasmid, aviral particle or a phage. In certain embodiments, the recombinantvector is an expression vector. The vectors may also include variousmarker genes which are useful in identifying cells containing suchvectors.

[0059] This invention also includes a cell comprising such a recombinantvector. The recombinant vectors described herein can be introduced intoa host cell, e.g., by transformation, transfection or infection.

[0060] This invention also includes a method for producing an LBPcomprising culturing such a cell under conditions that permit expressionof the LBP.

[0061] This invention also includes an isolated polypeptide comprising apolypeptide having the amino acid sequence as set forth in FIG. 1 (SEQID NO:1); FIG. 2 (SEQ ID NO:2); FIG. 3 (SEQ ID NO:3); FIG. 4 (SEQ IDNO:4); FIG. 5 (SEQ ID NO:5); FIG. 6 (SEQ ID NO:6); FIG. 7 (SEQ ID NO:7);FIG. 8 (SEQ ID NO:8) or FIG. 9 (SEQ ID NO:9); or a polypeptide which isat least about 80% identical, more preferably at least about 90%identical, more preferably yet at least about 95% identical, and mostpreferably at least about 98% identical to the above polypeptides, andwherein said polypeptide is capable of binding to LDL; or a biologicallyactive fragment of any of the above polypeptides wherein the fragment iscapable of binding to LDL. Differences in amino acids between the rabbitand human LBP-1, LBP-2 and LBP-3 genes are depicted in bold type in thefigures. The differences in the amino acid sequences between rabbit andhuman LBP-1, LBP-2 and LBP-3 are also specifically shown in FIGS. 19, 20and 21, respectively.

[0062] This invention also includes an isolated polypeptide comprising apolypeptide having amino acid residues 8-22 (SEQ ID NO:19), 8-33 (SEQ IDNO:20), 23-33 (SEQ ID NO:21) or 208-217 (SEQ ID NO:22) as set forth inFIG. 7 (SEQ ID NO:7); amino acid residues 14-43 (SEQ ID NO:23) or 38-43(SEQ ID NO:24) as set forth in FIG. 1 (SEQ ID NO:1) and FIG. 6 (SEQ IDNO:6); amino acid residues 105-120 (SEQ ID NO:25), 105-132 (SEQ IDNO:26), 121-132 (SEQ ID NO:27) or 211-220 (SEQ ID NO:28) as set forth inFIG. 2 (SEQ ID NO:2); amino acid residues 96-110 (SEQ ID NO:29) as setforth in FIG. 5 (SEQ ID NO:5); and amino acid residues 53-59 (SEQ IDNO:41) as set forth in FIG. 8 (SEQ ID NO:8); or a polypeptide which isat least about 80% identical, more preferably at least about 90%identical, more preferably yet at least about 95% identical, and mostpreferably at least about 98% identical to the above polypeptides, andwherein said polypeptide is capable of binding to LDL; or a biologicallyactive fragment of any of the above polypeptides wherein the fragment iscapable of binding to LDL.

[0063] The polypeptides of the invention are meant to include, e.g., anaturally purified product, a chemically synthesized product, and arecombinantly derived product.

[0064] The polypeptides can be used, e.g., to bind to LDL, therebyinhibiting formation of atherosclerotic plaques. The polypeptides canalso be used, e.g., in gene therapy, by expression of such polypeptidesin vivo. The polypeptides can also be used in pharmaceutical or vaccinecompositions. The polypeptides can also be used as immunogens to produceantibodies thereto, which in turn, can be used as antagonists to the LBPpolypeptides.

[0065] Without being bound by any theory, it is believed that the LBPsprovide the mechanism by which atherosclerosis is promoted through LDLoxidation. The LBPs are believed to be required in order for focal,irreversible LDL binding to occur at the arterial wall, and that suchbinding is a critical early event in atherosclerosis because it allowsthe time necessary for LDL to be changed from its native state to afully oxidized state. Since oxidized, but not native, LDL is a foreignprotein, macrophages ingest it, first becoming the foam cells of type Ilesions, and subsequently forming the fatty streaks of type II lesions.

[0066] This invention also includes a method for determining if ananimal is at risk for atherosclerosis. An animal is provided. An aspectof LBP metabolism or structure is evaluated in the animal. Anabnormality in the aspect of LBP metabolism or structure is diagnosticof being at risk for atherosclerosis.

[0067] By atherosclerosis is meant a disease or condition whichcomprises several stages which blend imperceptibly into each other,including irreversible binding of LDL, LDL oxidation, macrophagerecruitment, blockage of the artery and tissue death (infarction).

[0068] By animal is meant human as well as non-human animals. Non-humananimals include, e.g., mammals, birds, reptiles, amphibians, fish,insects and protozoa. Preferably, the non-human animal is a mammal,e.g., a rabbit, a rodent, e.g., a mouse, rat or guinea pig, a primate,e.g., a monkey, or a pig. An animal also includes transgenic non-humananimals. The term transgenic animal is meant to include an animal thathas gained new genetic information from the introduction of foreign DNA,i.e., partly or entirely heterologous DNA, into the DNA of its cells; orintroduction of a lesion, e.g., an in vitro induced mutation, e.g., adeletion or other chromosomal rearrangement into the DNA of its cells;or introduction of homologous DNA into the DNA of its cells in such away as to alter the genome of the cell into which the DNA is inserted,e.g., it is inserted at a location which differs from that of thenatural gene or its insertion results in a knockout or replacement ofthe homologous host gene or results in altered and/or regulatableexpression and/or metabolism of the gene. The animal may include atransgene in all of its cells including germ line cells, or in only oneor some of its cells. Transgenic animals of the invention can serve as amodel for studying atherosclerosis or for evaluating agents to treatatherosclerosis.

[0069] In certain embodiments, the determination for being at risk foratherosclerosis is done in a prenatal animal.

[0070] By LBP is meant a low density lipoprotein (LDL) binding proteinwhich is capable of binding LDL and methylated LDL. By methylated LDL ismeant that about 50% to about 90% of the lysine residues of LDL have amethyl group chemically attached. Methylated LDL is not recognized bypreviously reported cell surface receptors. See, e.g., Weisgraber etal., J. Biol. Chem. 253:9053-9062 (1978) In certain embodiments, the LBPis also capable of binding oxidized LDL. In certain preferredembodiments, the binding of LDL to an LBP is irreversible. In certainpreferred embodiments, the LBP does not transport the LDL to anyintracellular compartment. Examples of LBPs are LBP-1, LBP-2 and LBP-3described herein.

[0071] By LBP metabolism is meant any aspect of the production, release,expression, function, action, interaction or regulation of LBP. Themetabolism of LBP includes modifications, e.g., covalent or non-covalentmodifications, of LBP polypeptide. The metabolism of LBP includesmodifications, e.g., covalent or non-covalent modifications, that LBPinduces in other substances. The metabolism of LBP also includes changesin the distribution of LBP polypeptide, and changes LBP induces in thedistribution of other substances.

[0072] Any aspect of LBP metabolism can be evaluated. The methods usedare standard techniques known to those skilled in the art and can befound in standard references, e.g., Ausubel et al., ed., CurrentProtocols in Mol. Biology, New York: John Wiley & Sons, 1990; Kriegler,M., ed., Gene Transfer and Expression, Stockton Press, New York, N.Y.,1989; pDisplay gene expression system (Invitrogen, Carlsbad, Calif.).Preferred examples of LBP metabolism that can be evaluated include thebinding activity of LBP polypeptide to a binding molecule, e.g., LDL;the transactivation activity of LBP polypeptide on a target gene; thelevel of LBP protein; the level of LBP mRNA; the level of LBPmodifications, e.g., phosphorylation, glycosylation or acylation; or theeffect of LBP expression on transfected mammalian cell binding of LDL.

[0073] By binding molecule is meant any molecule to which LBP can bind,e.g., a nucleic acid, e.g., a DNA regulatory region, a protein, e.g.,LDL, a metabolite, a peptide mimetic, a non-peptide mimetic, anantibody, or any other type of ligand. In certain preferred embodiments,the aspect of LBP metabolism that is evaluated is the ability of LBP tobind to native LDL and/or methylated LDL and/or oxidized LDL. Binding toLDL can be shown, e.g., by antibodies against LDL, affinitychromatography, affinity coelectrophoresis (ACE) assays, or ELISAassays. See Examples. In other embodiments, it is the ability of LBP tobind to an arterial extracellular matrix stuctural component that isevaluated. Examples of such components include proteoglycans, e.g.,chondroitin sulfate proteoglycans and heparin sulfate proteoglycans;elastin; collagen; fibronectin; vitronectin; integrins; and relatedextracellular matrix molecules. Binding to arterial extracellular matrixstructural components can be shown by standard methods known to thoseskilled in the art, e.g., by ELISA assays. Primary antibodies to the LBPare then added, followed by an enzyme-conjugated secondary antibody tothe primary antibody, which produces a stable color in the presence ofan appropriate substrate, and color development on the plates ismeasured in a microtiter plate reader.

[0074] Transactivation of a target gene by LBP can be determined, e.g.,in a transient transfection assay in which the promoter of the targetgene is linked to a reporter gene, e.g., β-galactosidase or luciferase,and co-transfected with an LBP expression vector. Such evaluations canbe done in vitro or in vivo. Levels of LBP protein, mRNA orphosphorylation, can be measured, e.g., in a sample, e.g., a tissuesample, e.g., arterial wall, by standard methods known to those skilledin the art.

[0075] In certain embodiments, an aspect of LBP structure is evaluated,e.g., LBP gene structure or LBP protein structure. For example, primary,secondary or tertiary structures can be evaluated. For example, the DNAsequence of the gene is determined and/or the amino acid sequence of theprotein is determined. Standard cloning and sequencing methods can beused as are known to those skilled in the art. In certain embodiments,the binding activity of an antisense nucleic acid with the cellular LBPmRNA and/or genomic DNA is determined using standard methods known tothose skilled in the art so as to detect the presence or absence of thetarget mRNA or DNA sequences to which the antisense nucleic acid wouldnormally specifically bind.

[0076] The risk for atherosclerosis that is determined can be a reducedrisk or an increased risk as compared to a normal animal. For example,an abnormality which would give a reduced risk is an inactive LBPpolypeptide. An abnormality which would give an increased risk would be,e.g., an LBP polypeptide that has higher activity, e.g., LDL bindingactivity, than native LBP polypeptide.

[0077] The invention also includes a method for evaluating an agent foruse in treating atherosclerosis. A test cell, cell-free system or animalis provided. An agent is provided. The agent is administered to the testcell, cell-free system or animal in a therapeutically effective amount.The effect of the agent on an aspect of LBP metabolism or structure isevaluated. A change in the aspect of LBP metabolism or structure isindicative of the usefulness of the agent in treating atherosclerosis.

[0078] In certain embodiments, the method employs two phases forevaluating an agent for use in treating atherosclerosis, an initial invitro phase and then an in vivo phase. The agent is administered to thetest cell or cell-free system in vitro, and if a change in an aspect ofLBP metabolism occurs, then the agent is further administered to a testanimal in a therapeutically effective amount and evaluated in vivo foran effect of the agent on an aspect of LBP metabolism.

[0079] By cell is meant a cell or a group of cells, or a cell that ispart of an animal. The cell can be a human or non-human cell. Cell isalso meant to include a transgenic cell. The cell can be obtained, e.g.,from a culture or from an animal. Animals are meant to include, e.g.,natural animals and non-human transgenic animals. In certainembodiments, the transgenic cell or non-human transgenic animal has anLBP transgene, or fragment or analog thereof. In certain embodiments,the transgenic cell or non-human transgenic animal has a knockout forthe LBP gene.

[0080] The test cell, cell-free system or animal can have a wild typepattern or a non-wild type pattern of LBP metabolism. A non-wild typepattern of LBP metabolism can result, e.g., from under-expression,over-expression, no expression, or a temporal, site or distributionchange. Such a non-wild type pattern can result, e.g., from one or moremutations in the LBP gene, in a binding molecule gene, a regulatorygene, or in any other gene which directly or indirectly affects LBPmetabolism. A mutation is meant to include, e.g., an alteration, e.g.,in gross or fine structure, in a nucleic acid. Examples include singlebase pair alterations, e.g., missense or nonsense mutations,frameshifts, deletions, insertions and translocations. Mutations can bedominant or recessive. Mutations can be homozygous or heterozygous.Preferably, an aspect of LBP-1, LBP-2 or LBP-3 metabolism is evaluated.

[0081] An agent is meant to include, e.g., any substance, e.g., ananti-atherosclerosis drug. The agent of this invention preferably canchange an aspect of LBP metabolism. Such change can be the result of anyof a variety of events, including, e.g., preventing or reducinginteraction between LBP and a binding molecule, e.g., LDL or an arterialextracellular matrix structural component; inactivating LBP and/or thebinding molecule, e.g., by cleavage or other modification; altering theaffinity of LBP and the binding molecule for each other; diluting outLBP and/or the binding molecule; preventing expression of LBP and/or thebinding molecule; reducing synthesis of LBP and/or the binding molecule;synthesizing an abnormal LBP and/or binding molecule; synthesizing analternatively spliced LBP and/or binding molecule; preventing orreducing proper conformational folding of LBP and/or the bindingmolecule; modulating the binding properties of LBP and/or the bindingmolecule; interfering with signals that are required to activate ordeactivate LBP and/or the binding molecule; activating or deactivatingLBP and/or the binding molecule in such a way as to prevent binding; orinterfering with other receptors, ligands or other molecules which arerequired for the normal synthesis or functioning of LBP and/or thebinding molecule. For example, the agent can block the binding site onLDL for LBPs expressed focally in the arterial wall extracellularmatrix, or it could block the binding site on an LBP for LDL, or itcould be bifunctional, i.e., it could block both binding sites.

[0082] Examples of agents include LBP polypeptide, e.g., LBP-1, LBP-2 orLBP-3, or a biologically active fragment or analog thereof; a nucleicacid encoding LBP polypeptide or a biologically active fragment oranalog thereof; a nucleic acid encoding an LBP regulatory sequence or abiologically active fragment or analog thereof; a binding molecule forLBP polypeptide; a binding molecule for LBP nucleic acid, the LBPnucleic acid being, e.g., a nucleic acid comprising a regulatory regionfor LBP or a nucleic acid comprising a structural region for LBP or abiologically active fragment of LBP; an antisense nucleic acid; amimetic of LBP or a binding molecule; an antibody for LBP or a bindingmolecule; a metabolite; or an inhibitory carbohydrate or glycoprotein.In certain embodiments, the agent is an antagonist, agonist or superagonist.

[0083] Knowledge of the existence of the sequence of the LBPs allows asearch for natural or artificial ligands to regulate LDL levels in thetreatment of atherosclerosis. In certain embodiments, the agent is anatural ligand for LBP. In certain embodiments, the agent is anartificial ligand for LBP.

[0084] By analog is meant a compound that differs from naturallyoccurring LBP in amino acid sequence or in ways that do not involvesequence, or both. Analogs of the invention generally exhibit at leastabout 80% homology, preferably at least about 90% homology, morepreferably yet at least about 95% homology, and most preferably at leastabout 98% homology, with substantially the entire sequence of anaturally occurring LBP sequence, preferably with a segment of about 100amino acid residues, more preferably with a segment of about 50 aminoacid residues, more preferably yet with a segment of about 30 amino acidresidues, more preferably yet with a segment of about 20 amino acidresidues, more preferably yet with a segment of about 10 amino acidresidues, more preferably yet with a segment of about 5 amino acidresidues, more preferably yet with a segment of about 4 amino acidresidues, more preferably yet with a segment of about 3 amino acidresidues, and most preferably with a segment of about 2 amino acidresidues. Non-sequence modifications include, e.g., in vivo or in vitrochemical derivatizations of LBP. Non-sequence modifications include,e.g., changes in phosphorylation, acetylation, methylation,carboxylation, or glycosylation. Methods for making such modificationsare known to those skilled in the art. For example, phosphorylation canbe modified by exposing LBP to phosphorylation-altering enzymes, e.g.,kinases or phosphatases.

[0085] Preferred analogs include LBP or biologically active fragmentsthereof whose sequences differ from the wild-type sequence by one ormore conservative amino acid substitutions or by one or morenon-conservative amino acid substitutions, deletions, or insertionswhich do not abolish LBP biological activity. Conservative substitutionstypically include the substitution of one amino acid for another withsimilar characteristics, e.g., substitutions within the followinggroups: valine, glycine; glycine, alanine; valine, isoleucine, leucine;aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine;lysine, arginine; and phenylalanine, tyrosine. Other examples ofconservative substitutions are shown in Table 1. TABLE 1 CONSERVATIVEAMINO ACID SUBSTITUTIONS For Amino Acid Code Replace with any of AlanineA D-Ala, Gly, beta-Ala, L-Cys, D-Cys Arginine R D-Arg, Lys, D-Lys,homo-Arg, D-homo-Arg, Met, Ile, D-Met, D-Ile, Orn, D-Orn, L-NMMA, L-NAMEAsparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln Aspartic Acid DD-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine C D-Cys, S-Me-Cys,Met, D-Met, Thr, D-Thr Glutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp,D-Asp Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln GlycineG Ala, D-Ala, Pro, D-Pro, β-Ala Asp Histidine H D-His Isoleucine ID-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu, Val, D-Val,Leu, D-Leu, Met, D-Met Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg,Met, D-Met, Ile, D-Ile, Orn, D-Orn Methionine M D-Met, S-Me-Cys, Ile,D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine F D-Phe, Tyr, D-Thr, L-Dopa,His, D-His, Trp, D-Trp, Trans-3,4, or 5- phenylproline, cis-3,4, or 5-phenylproproline Proline P D-Pro, L-I-thioazolidine-4-carboxylic acid,D-or L-1-oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr,allo-Thr, Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys Threonine T D-Thr,Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val, D-ValTryptophan W D-Trp, Phe, D-Phe, Tyr, D-Tyr Tyrosine Y D-Tyr, Phe, D-Phe,L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

[0086] Amino acid sequence variants of a protein can be prepared by anyof a variety of methods known to those skilled in the art. For example,random mutagenesis of DNA which encodes a protein or a particular domainor region of a protein can be used, e.g., PCR mutagenesis (using, e.g.,reduced Taq polymerase fidelity to introduce random mutations into acloned fragment of DNA; Leung et al., BioTechnique 1:11-15 (1989)), orsaturation mutagenesis (by, e.g., chemical treatment or irradiation ofsingle-stranded DNA in vitro, and synthesis of a complementary DNAstrand; Mayers et al., Science 229:242 (1985)). Random mutagenesis canalso be accomplished by, e.g., degenerate oligonucleotide generation(using, e.g., an automatic DNA synthesizer to chemically synthesizedegenerate sequences; Narang, Tetrahedron 39:3 (1983); Itakura et al.,Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. A. G.Walton, Amsterdam: Elsevier, pp. 273-289 (1981)). Non-random or directedmutagenesis can be used to provide specific sequences or mutations inspecific regions. These techniques can be used to create variants whichinclude, e.g., deletions, insertions, or substitutions, of residues ofthe known amino acid sequence of a protein. The sites for mutation canbe modified individually or in series, e.g., by (i) substituting firstwith conserved amino acids and then with more radical choices dependingupon results achieved, (ii) deleting the target residue, (iii) insertingresidues of the same or a different class adjacent to the located site,or (iv) combinations of the above. For example, analogs can be made byin vitro DNA sequence modifications of the sequences of FIGS. 10-18 (SEQID NOS:10-18). For example, in vitro mutagenesis can be used to convertany of these DNA sequences into a sequence which encodes an analog inwhich one or more amino acid residues has undergone a replacement, e.g.,a conservative replacement as described in Table 1.

[0087] Methods for identifying desirable mutations include, e.g.,alanine scanning mutagenesis (Cunningham and Wells, Science244:1081-1085 (1989)), oligonucleotide-mediated mutagenesis (Adelman etal., DNA 2:183 (1983)); cassette mutagenesis (Wells et al., Gene 34:315(1985)), combinatorial mutagenesis, and phage display libraries (Ladneret al., PCT International Appln. No. WO88/06630). The LBP analogs can betested, e.g., for their ability to bind to LDL and/or to an arterialextracellular matrix component, as described herein.

[0088] Other analogs within the invention include, e.g., those withmodifications which increase peptide stability. Such analogs maycontain, e.g., one or more non-peptide bonds (which replace the peptidebonds) in the peptide sequence. Also included are, e.g.: analogs thatinclude residues other than naturally occurring L-amino acids, e.g.,D-amino acids or non-naturally occurring or synthetic amino acids, e.g.,β or γ amino acids; and cyclic analogs.

[0089] Analogs are also meant to include peptides in which structuralmodifications have been introduced into the peptide backbone so as tomake the peptide non-hydrolyzable. Such peptides are particularly usefulfor oral administration, as they are not digested. Peptide backbonemodifications include, e.g., modifications of the amide nitrogen, theα-carbon, the amide carbonyl, or the amide bond, and modificationsinvolving extensions, deletions or backbone crosslinks. For example, thebackbone can be modified by substitution of a sulfoxide for thecarbonyl, by reversing the peptide bond, or by substituting a methylenefor the carbonyl group. Such modifications can be made by standardprocedures known to those skilled in the art. See, e.g., Spatola, A. F.,“Peptide Backbone Modifications: A Structure-Activity Analysis ofPeptides Containing Amide Bond Surrogates, Conformational Constraints,and Related Backbone Replacements,” in Chemistry and Biochemistry ofAmino Acids, Peptides and Proteins, Vol. 7, pp. 267-357, B. Weinstein(ed.), Marcel Dekker, Inc., New York (1983).

[0090] An analog is also meant to include polypeptides in which one ormore of the amino acid residues include a substituent group, orpolypeptides which are fused with another compound, e.g., a compound toincrease the half-life of the polypeptide, e.g., polyethylene glycol.

[0091] By fragment is meant some portion of the naturally occurring LBPpolypeptide. Preferably, the fragment is at least about 100 amino acidresidues, more preferably at least about 50 amino acid residues, morepreferably yet at least about 30 amino acid residues, more preferablyyet at least about 20 amino acid residues, more preferably yet at leastabout 5 amino acid residues, more preferably yet at least about 4 aminoacid residues, more preferably yet at least about 3 amino acid residues,and most preferably at least about 2 amino acid residues in length.Fragments include, e.g., truncated secreted forms, proteolyticfragments, splicing fragments, other fragments, and chimeric constructsbetween at least a portion of the relevant gene, e.g., LBP-1, LBP-2 orLBP-3, and another molecule. Fragments of LBP can be generated bymethods known to those skilled in the art. In certain embodiments, thefragment is biologically active. The ability of a candidate fragment toexhibit a biological activity of LBP can be assessed by methods known tothose skilled in the art. For example, LBP fragments can be tested fortheir ability to bind to LDL and/or to an arterial extracellular matrixstructural component, as described herein. Also included are LBPfragments containing residues that are not required for biologicalactivity of the fragment or that result from alternative mRNA splicingor alternative protein processing events.

[0092] Fragments of a protein can be produced by any of a variety ofmethods known to those skilled in the art, e.g., recombinantly, byproteolytic digestion, or by chemical synthesis. Internal or terminalfragments of a polypeptide can be generated by removing one or morenucleotides from one end (for a terminal fragment) or both ends (for aninternal fragment) of a nucleic acid which encodes the polypeptide.Expression of the mutagenized DNA produces polypeptide fragments.Digestion with “end-nibbling” endonucleases can thus generate DNAs whichencode an array of fragments. DNAs which encode fragments of a proteincan also be generated, e.g., by random shearing, restriction digestionor a combination of the above-discussed methods. For example, fragmentsof LBP can be made by expressing LBP DNA which has been manipulated invitro to encode the desired fragment, e.g., by restriction digestion ofany of the DNA sequences of FIGS. 10-18 (SEQ ID NOS:10-18).

[0093] Fragments can also be chemically synthesized using techniquesknown in the art, e.g., conventional Merrifield solid phase f-Moc ort-Boc chemistry. For example, peptides of the present invention can bearbitrarily divided into fragments of desired length with no overlap ofthe fragments, or divided into overlapping fragments of a desiredlength.

[0094] An LBP or a biologically active fragment or analog thereof, or abinding molecule or a biologically active fragment or analog thereof,can, e.g., compete with its cognate molecule for the binding site on thecomplementary molecule, and thereby reduce or eliminate binding betweenLBP and the cellular binding molecule. LBP or a binding molecule can beobtained, e.g., from purification or secretion of naturally occurringLBP or binding molecule, from recombinant LBP or binding molecule, orfrom synthesized LBP or binding molecule.

[0095] Therefore, methods for generating analogs and fragments andtesting them for activity are known to those skilled in the art.

[0096] An agent can also be a nucleic acid used as an antisensemolecule. Antisense therapy is meant to include, e.g., administration orin situ generation of oligonucleotides or their derivatives whichspecifically hybridize, e.g., bind, under cellular conditions, with thecellular mRNA and/or genomic DNA encoding an LBP polypeptide, or mutantthereof, so as to inhibit expression of the encoded protein, e.g., byinhibiting transcription and/or translation. The binding may be byconventional base pair complementarity, or, for example, in the case ofbinding to DNA duplexes, through specific interactions in the majorgroove of the double helix.

[0097] In certain embodiments, the antisense construct binds to anaturally-occurring sequence of an LBP gene which, e.g., is involved inexpression of the gene. These sequences include, e.g., promoter, startcodons, stop codons, and RNA polymerase binding sites.

[0098] In other embodiments, the antisense construct binds to anucleotide sequence which is not present in the wild type gene. Forexample, the antisense construct can bind to a region of an LBP genewhich contains an insertion of an exogenous, non-wild type sequence.Alternatively, the antisense construct can bind to a region of an LBPgene which has undergone a deletion, thereby bringing two regions of thegene together which are not normally positioned together and which,together, create a non-wild type sequence. When administered in vivo toa subject, antisense constructs which bind to non-wild type sequencesprovide the advantage of inhibiting the expression of a mutant LBP gene,without inhibiting expression of any wild type LBP gene.

[0099] An antisense construct of the present invention can be delivered,e.g., as an expression plasmid which, when transcribed in the cell,produces RNA which is complementary to at least a unique portion of thecellular mRNA which encodes an LBP polypeptide. An alternative is thatthe antisense construct is an oligonucleotide which is generated ex vivoand which, when introduced into the cell causes inhibition of expressionby hybridizing with the mRNA (duplexing) and/or genomic sequences(triplexing) of an LBP gene. Such oligonucleotides are preferablymodified oligonucleotides which are resistant to endogenous nucleases,e.g. exonucleases and/or endonucleases, and are therefore stable invivo. Exemplary nucleic acid molecules for use as antisenseoligonucleotides are phosphoramidate, phosphothioate,phosphorodithioates and methylphosphonate analogs of DNA and peptidenucleic acids (PNA). (See also U.S. Pat. Nos. 5,176,996; 5,264,564; and5,256,775). Additionally, general approaches to constructing oligomersuseful in antisense therapy have been reviewed. (See, e.g., Van der Krolet al., Biotechniques 6:958-976, (1988); Stein et al., Cancer Res.48:2659-2668 (1988)).

[0100] By mimetic is meant a molecule which resembles in shape and/orcharge distribution LBP or a binding molecule. The mimetic can be apeptide or a non-peptide. Mimetics can act as therapeutic agents becausethey can, e.g., competitively inhibit binding of LBP to a bindingmolecule. By employing, e.g., scanning mutagenesis, e.g., alaninescanning mutagenesis, linker scanning mutagenesis or saturationmutagenesis, to map the amino acid residues of a particular LBPpolypeptide involved in binding a binding molecule, peptide mimetics,e.g., diazepine or isoquinoline derivatives, can be generated whichmimic those residues in binding to a binding molecule, and whichtherefore can inhibit binding of the LBP to a binding molecule andthereby interfere with the function of LBP. Non-hydrolyzable peptideanalogs of such residues can be generated using, e.g., benzodiazepine(see, e.g., Freidinger et al., in Peptides: Chemistry and Biology, G. R.Marshall ed., ESCOM Publisher: Leiden, Netherlands (1988)); azepine(see, e.g., Huffman et al., in Peptides: Chemistry and Biology, G. R.Marshall ed., ESCOM Publisher: Leiden, Netherlands (1988)); substitutedgamma lactam rings (see, e.g., Garvey et al., in Peptides: Chemistry andBiology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands(1988)); keto-methylene pseudopeptides (see, e.g., Ewenson et al., J.Med. Chem. 29:295 (1986); Ewenson et al., in Peptides: Structure andFunction (Proceedings of the 9th American Peptide Symposium) PierceChemical Co. Rockland, Ill. (1985)); β-turn dipeptide cores (see, e.g.,Nagai et al., Tetrahedron Lett. 26:647 (1985); Sato et al., J. Chem.Soc. Perkin Trans. 1:1231 (1986)); or β-aminoalcohols (see, e.g., Gordonet al., Biochem. Biophys. Res. Commun. 126:419 (1985); Dann et al.,Biochem. Biophys. Res. Commun. 134:71 (1986)).

[0101] Antibodies are meant to include antibodies against any moietythat directly or indirectly affects LBP metabolism. The antibodies canbe directed against, e.g., LBP or a binding molecule, or a subunit orfragment thereof. For example, antibodies include anti-LBP-1, LBP-2 orLBP-3 antibodies; and anti-binding molecule antibodies. Antibodyfragments are meant to include, e.g., Fab fragments, Fab′ fragments,F(ab′)₂ fragments, F(v) fragments, heavy chain monomers, heavy chaindimers, heavy chain trimers, light chain monomers, light chain dimers,light chain trimers, dimers consisting of one heavy and one light chain,and peptides that mimic the activity of the anti-LBP or anti-bindingmolecule antibodies. For example, Fab₂′ fragments of the inhibitoryantibody can be generated through, e.g., enzymatic cleavage. Bothpolyclonal and monoclonal antibodies can be used in this invention.Preferably, monoclonal antibodies are used. Natural antibodies,recombinant antibodies or chimeric-antibodies, e.g., humanizedantibodies, are included in this invention. Preferably, humanizedantibodies are used when the subject is a human. Most preferably, theantibodies have a constant region derived from a human antibody and avariable region derived from an inhibitory mouse monoclonal antibody.Production of polyclonal antibodies to LBP is described in Example 6.Monoclonal and humanized antibodies are generated by standard methodsknown to those skilled in the art. Monoclonal antibodies can beproduced, e.g., by any technique which provides antibodies produced bycontinuous cell lines cultures. Examples include the hybridoma technique(Kohler and Milstein, Nature 256:495-497 (1975), the trioma technique,the human B-cell hybridoma technique (Kozbor et al., Immunology Today4:72 (1983)), and the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al., in Monoclonal Antibodies and CancerTherapy, A.R. Liss, Inc., pp. 77-96 (1985)). Preferably, humanizedantibodies are raised through conventional production and harvestingtechniques (Berkower, I., Curr. Opin. Biotechnol. 7:622-628 (1996);Ramharayan and Skaletsky, Am. Biotechnol. Lab 13:26-28 (1995)). Incertain preferred embodiments, the antibodies are raised against theLBP, preferably the LDL-binding site, and the Fab fragments produced.These antibodies, or fragments derived therefrom, can be used, e.g., toblock the LDL-binding sites on the LBP molecules.

[0102] Agents also include inhibitors of a molecule that are requiredfor synthesis, post-translational modification, or functioning of LBPand/or a binding molecule, or activators of a molecule that inhibits thesynthesis or functioning of LBP and/or the binding molecule. Agentsinclude, e.g., cytokines, chemokines, growth factors, hormones,signaling components, kinases, phosphatases, homeobox proteins,transcription factors, editing factors, translation factors andpost-translation factors or enzymes. Agents are also meant to includeionizing radiation, non-ionizing radiation, ultrasound and toxic agentswhich can, e.g., at least partially inactivate or destroy LBP and/or thebinding molecule.

[0103] An agent is also meant to include an agent which is not entirelyLBP specific. For example, an agent may alter other genes or proteinsrelated to arterial plaque formation. Such overlapping specificity mayprovide additional therapeutic advantage.

[0104] The invention also includes the agent so identified as beinguseful in treating atherosclerosis.

[0105] The invention also includes a method for evaluating an agent forthe ability to alter the binding of LBP polypeptide to a bindingmolecule. An agent is provided. An LBP polypeptide is provided. Abinding molecule is provided. The agent, LBP polypeptide and bindingmolecule are combined. The formation of a complex comprising the LBPpolypeptide and binding molecule is detected. An alteration in theformation of the complex in the presence of the agent as compared to inthe absence of the agent is indicative of the agent altering the bindingof the LBP polypeptide to the binding molecule.

[0106] In preferred embodiments, the LBP polypeptide is LBP-1, LBP-2 orLBP-3. Examples of a binding molecule include native LDL, modified LDL,e.g., methylated LDL or oxidized LDL, and arterial extracellular matrixstructural components.

[0107] Altering the binding includes, e.g., inhibiting or promoting thebinding. The efficacy of the agent can be assessed, e.g., by generatingdose response curves from data obtained using various concentrations ofthe agent. Methods for determining formation of a complex are standardand are known to those skilled in the art, e.g., affinitycoelectrophoresis (ACE) assays or ELISA assays as described herein.

[0108] The invention also includes the agent so identified as being ableto alter the binding of an LBP polypeptide to a binding molecule.

[0109] The invention also includes a method for evaluating an agent forthe ability to bind to an LBP polypeptide. An agent is provided. An LBPpolypeptide is provided. The agent is contacted with the LBPpolypeptide. The ability of the agent to bind to the LBP polypeptide isevaluated. Preferably, the LBP polypeptide is LBP-1, LBP-2 or LBP-3.Binding can be determined, e.g., by measuring formation of a complex bystandard methods known to those skilled in the art, e.g., affinitycoelectrophoresis (ACE) assays or ELISA assays as described herein.

[0110] The invention also includes the agent so identified as being ableto bind to LBP polypeptide.

[0111] The invention also includes a method for evaluating an agent forthe ability to bind to a nucleic acid encoding an LBP regulatorysequence. An agent is provided. A nucleic acid encoding an LBPregulatory sequence is provided. The agent is contacted with the nucleicacid. The ability of the agent to bind to the nucleic acid is evaluated.Preferably, the LBP regulatory sequence is an LBP-1, LBP-2 or LBP-3regulatory sequence. Binding can be determined, e.g., by measuringformation of a complex by standard methods known to those skilled in theart, e.g., DNA mobility shift assays, DNase I footprint analysis(Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley& Sons, New York, N.Y., (1989)).

[0112] The invention also includes the agent so identified as being ableto bind to a nucleic acid encoding an LBP regulatory sequence.

[0113] The invention also includes a method for treating atherosclerosisin an animal. An animal in need of treatment for atherosclerosis isprovided. An agent capable of altering an aspect of LBP structure ormetabolism is provided. The agent is administered to the animal in atherapeutically effective amount such that treatment of theatherosclerosis occurs.

[0114] In certain preferred embodiments, the agent is an LBPpolypeptide, e.g., LBP-1, LBP-2 or LBP-3, or a biologically activefragment or analog thereof. The agent can be, e.g., the polypeptide asset forth in SEQ ID NOS:1-9. Preferably, the agent is a polypeptide ofno more than about 100 amino acid residues in length, more preferably ofno more than about 50 amino acid residues, more preferably yet of nomore than about 30 amino acid residues, more preferably yet of no morethan about 20 amino acid residues, more preferably yet of no more thanabout 10 amino acid residues, more preferably yet of no more than about5 amino acid residues, more preferably yet of no more than about 4 aminoacid residues, more preferably yet of no more than about 3 amino acidresidues, and most preferably of no more than about 2 amino acidresidues. Preferably, the polypeptide includes at least about 20% acidicamino acid residues, more preferably yet at least about 40% acidic aminoacid residues, more preferably yet at least about 60% acidic amino acidresidues, more preferably yet at least about 80% acidic amino acidresidues, more preferably yet at least about 90% acidic amino acidresidues, more preferably yet at least about 95% acidic amino acidresidues, and most preferably at least about 98% acidic amino acidresidues. Acidic amino acid residues include aspartic acid and glutamicacid. An example of such an LBP polypeptide is BHF-1, which is a 20amino acid length fragment of human or rabbit LBP-1 which contains aminoacid residues 14 through 33. See FIG. 9 (SEQ ID NO:9). 45% of the aminoacid residues of BHF-1 are acidic. The invention also includesbiologically active fragments and analogs of BHF-1.

[0115] Other preferred acidic regions from the LBPs are amino acidresidues 8 through 22 (SEQ ID NO:19), 8 through 33 (SEQ ID NO:20), 23through 33 (SEQ ID NO:21), and 208 through 217 (SEQ ID NO:22) of humanLBP-2 as depicted in FIG. 7 (SEQ. ID NO:7); amino acid residues 14through 43 (SEQ ID NO:23) and 38 through 43 (SEQ ID NO:24) of rabbit orhuman LBP-1 as depicted in FIG. 1 (SEQ ID NO:1) and FIG. 6 (SEQ IDNO:6); amino acid residues 105 through 120 (SEQ ID NO:25), 105 through132 (SEQ ID NO:26), 121 through 132 (SEQ ID NO:27), and 211 through 220(SEQ ID NO:28) of rabbit LBP-2 as depicted in FIG. 2 (SEQ ID NO:2);amino acid residues 96 through 110 (SEQ ID NO:29) of rabbit LBP-3 asdepicted in FIG. 5 (SEQ ID NO:5); and amino acid residues 53-59 (SEQ IDNO:41) of human LBP-3 as depicted in FIG. 8 (SEQ ID NO:8). The inventionis also meant to include biologically active fragments and analogs ofany of these polypeptides.

[0116] Other examples of agents include homopolymers and heteropolymersof any amino acid or amino acid analog. In certain preferredembodiments, the agent is a homopolymer of an acidic amino acid oranalog thereof. In certain embodiments, the agent is a heteropolymer ofone or more acidic amino acids and one or more other amino acids, oranalogs thereof. For example, agents include poly(glu), poly(asp),poly(glu asp), poly(glu N), poly(asp N) and poly(glu asp N). By N ismeant any amino acid, or analog thereof, other than glu or asp. Bypoly(glu asp) is meant all permutations of glu and asp for a givenlength peptide. A preferred peptide is poly(glu) of no more than about10 amino acids in length, preferably about 7 amino acids in length.

[0117] In certain preferred embodiments, the agent is an LBP nucleicacid or a biologically active fragment or analog thereof, e.g., anucleic acid encoding LBP-1, LBP-2 or LBP-3 polypeptide, or abiologically active fragment or analog thereof. The agent can be, e.g.,a nucleic acid comprising a nucleotide sequence as set forth in SEQ IDNOS:10-18. In other embodiments, the agent is an antisense molecule,e.g., one which can bind to an LBP gene sequence.

[0118] Treating is meant to include, e.g., preventing, treating,reducing the symptoms of, or curing the atherosclerosis. Administrationof the agent can be accomplished by any method which allows the agent toreach the target cells. These methods include, e.g., injection,deposition, implantation, suppositories, oral ingestion, inhalation,topical administration, or any other method of administration whereaccess to the target cells by the agent is obtained. Injections can be,e.g., intravenous, intradermal, subcutaneous, intramuscular orintraperitoneal. Implantation includes inserting implantable drugdelivery systems, e.g., microspheres, hydrogels, polymeric reservoirs,cholesterol matrices, polymeric systems, e.g., matrix erosion and/ordiffusion systems and non-polymeric systems, e.g., compressed, fused orpartially fused pellets. Suppositories include glycerin suppositories.Oral ingestion doses can be enterically coated. Inhalation includesadministering the agent with an aerosol in an inhalator, either alone orattached to a carrier that can be absorbed.

[0119] Administration of the agent can be alone or in combination withother therapeutic agents. In certain embodiments, the agent can becombined with a suitable carrier, incorporated into a liposome, orincorporated into a polymer release system.

[0120] In certain embodiments of the invention, the administration canbe designed so as to result in sequential exposures to the agent oversome time period, e.g., hours, days, weeks, months or years. This can beaccomplished by repeated administrations of the agent by one of themethods described above, or alternatively, by a controlled releasedelivery system in which the agent is delivered to the animal over aprolonged period without repeated administrations. By a controlledrelease delivery system is meant that total release of the agent doesnot occur immediately upon administration, but rather is delayed forsome time. Release can occur in bursts or it can occur gradually andcontinuously. Administration of such a system can be, e.g., by longacting oral dosage forms, bolus injections, transdermal patches orsubcutaneous implants.

[0121] Examples of systems in which release occurs in bursts include,e.g., systems in which the agent is entrapped in liposomes which areencapsulated in a polymer matrix, the liposomes being sensitive to aspecific stimulus, e.g., temperature, pH, light, magnetic field, or adegrading enzyme, and systems in which the agent is encapsulated by anionically-coated microcapsule with a microcapsule core-degrading enzyme.Examples of systems in which release of the agent is gradual andcontinuous include, e.g., erosional systems in which the agent iscontained in a form within a matrix, and diffusional systems in whichthe agent permeates at a controlled rate, e.g., through a polymer. Suchsustained release systems can be, e.g., in the form of pellets orcapsules.

[0122] The agent can be suspended in a liquid, e.g., in dissolved formor colloidal form. The liquid can be a solvent, partial solvent ornon-solvent. In many cases water or an organic liquid can be used.

[0123] The agent can be administered prior to or subsequent to theappearance of atherosclerosis symptoms. In certain embodiments, theagent is administered to patients with familial histories ofatherosclerosis, or who have phenotypes that may indicate apredisposition to atherosclerosis, or who have been diagnosed as havinga genotype which predisposes the patient to atherosclerosis, or who haveother risk factors, e.g., hypercholesterolemia, hypertension or smoking.

[0124] The agent is administered to the animal in a therapeuticallyeffective amount. By therapeutically effective amount is meant thatamount which is capable of at least partially preventing or reversingatherosclerosis. A therapeutically effective amount can be determined onan individual basis and will be based, at least in part, onconsideration of the species of animal, the animal's size, the animal'sage, the agent used, the type of delivery system used, the time ofadministration relative to the onset of atherosclerosis symptoms, andwhether a single, multiple, or controlled release dose regimen isemployed. A therapeutically effective amount can be determined by one ofordinary skill in the art employing such factors and using no more thanroutine experimentation.

[0125] Preferably, the concentration of the agent is at a dose of about0.1 to about 1000 mg/kg body weight/day, more preferably at about 0.1 toabout 500 mg/kg/day, more preferably yet at about 0.1 to about 100mg/kg/day, and most preferably at about 0.1 to about 5 mg/kg/day. Thespecific concentration partially depends upon the particular agent used,as some are more effective than others. The dosage concentration of theagent that is actually administered is dependent at least in part uponthe final concentration that is desired at the site of action, themethod of administration, the efficacy of the particular agent, thelongevity of the particular agent, and the timing of administrationrelative to the onset of the atherosclerosis symptoms. Preferably, thedosage form is such that it does not substantially deleteriously affectthe animal. The dosage can be determined by one of ordinary skill in theart employing such factors and using no more than routineexperimentation.

[0126] In certain embodiments, various gene constructs can be used aspart of a gene therapy protocol to deliver nucleic acids encoding anagent, e.g., either an agonistic or antagonistic form of an LBPpolypeptide. For example, expression vectors can be used for in vivotransfection and expression of an LBP polypeptide in particular celltypes so as to reconstitute the function of, or alternatively, abrogatethe function of, LBP polypeptide in a cell in which non-wild type LBP isexpressed. Expression constructs of the LBP polypeptide, and mutantsthereof, may be administered in any biologically effective carrier, e.g.any formulation or composition capable of effectively delivering the LBPgene to cells in vivo. Approaches include, e.g., insertion of thesubject gene in viral vectors including, e.g., recombinant retroviruses,adenovirus, adeno-associated virus, and herpes simplex virus-1, orrecombinant bacterial or eukaryotic plasmids. Viral vectors infect ortransduce cells directly; plasmid DNA can be delivered with the help of,for example, cationic liposomes (lipofectin™ (Life Technologies, Inc.,Gaithersburg, Md.) or derivatized (e.g. antibody conjugated), polylysineconjugates, gramacidin S, artificial viral envelopes or other suchintracellular carriers, as well as direct injection of the geneconstruct or Ca₃(PO₄)₂ precipitation carried out in vivo. Theabove-described methods are known to those skilled in the art and can beperformed without undue experimentation. Since transduction ofappropriate target cells represents the critical first step in genetherapy, choice of the particular gene delivery system will depend onsuch factors as the phenotype of the intended target and the route ofadministration, e.g., locally or systemically. Administration can bedirected to one or more cell types, and to one or more cells within acell type, so as to be therapeutically effective, by methods that areknown to those skilled in the art. In a preferred embodiment, the agentis administered to arterial wall cells of the animal. For example, agenetically engineered LBP gene is administered to arterial wall cells.In certain embodiments, administration is done in a prenatal animal orembryonic cell. It will be recognized that the particular gene constructprovided for in in vivo transduction of LBP expression is also usefulfor in vitro transduction of cells, such as for use in the diagnosticassays described herein.

[0127] In certain embodiments, therapy of atherosclerosis is performedwith antisense nucleotide analogs of the genes which code for the LBPs.Preferably, the antisense nucleotides have non-hydrolyzable “backbones,”e.g., phosphorothioates, phosphorodithioates or methylphosphonates. Thenucleoside base sequence is complementary to the sequence of a portionof the gene coding for, e.g., LBP-1, 2 or 3. Such a sequence might be,e.g., ATTGGC if the gene sequence for the LBP is TAACCG. One embodimentof such therapy would be incorporation of an antisense analog of aportion of one of the LBP genes in a slow-release medium, e.g.,polyvinyl alcohol, which is administered, e.g., by subcutaneousinjection, so as to release the antisense nucleotide analog over aperiod of weeks or months. In another embodiment, the antisense analogis incorporated into a polymeric matrix, e.g., polyvinyl alcohol, suchthat the gel can be applied locally to an injured arterial wall toinhibit LBP synthesis and prevent LDL accumulation, e.g., afterangioplasty or atherectomy.

[0128] The invention also includes a method for treating an animal atrisk for atherosclerosis. An animal at risk for atherosclerosis isprovided. An agent capable of altering an aspect of LBP structure ormetabolism is provided. The agent is administered to the animal in atherapeutically effective amount such that treatment of the animaloccurs. Being at risk for atherosclerosis can result from, e.g., afamily history of atherosclerosis, a genotype which predisposes toatherosclerosis, or phenotypic symptoms which predispose toatherosclerosis, e.g., having hypercholesterolemia, hypertension orsmoking.

[0129] The invention also includes a method for treating a cell havingan abnormality in structure or metabolism of LBP. A cell having anabnormality in structure or metabolism of LBP is provided. An agentcapable of altering an aspect of LBP structure or metabolism isprovided. The agent is administered to the cell in a therapeuticallyeffective amount such that treatment of the cell occurs.

[0130] In certain embodiments, the cell is obtained from a cell cultureor tissue culture or an embryo fibroblast. The cell can be, e.g., partof an animal, e.g., a natural animal or a non-human transgenic animal.Preferably, the LBP is LBP-1, LBP-2 or LBP-3.

[0131] The invention also includes a pharmaceutical composition fortreating atherosclerosis in an animal comprising a therapeuticallyeffective amount of an agent, the agent being capable of altering anaspect of LBP metabolism or structure in the animal so as to result intreatment of the atherosclerosis, and a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers include, e.g., saline,liposomes and lipid emulsions.

[0132] In certain preferred embodiments, the agent of the pharmaceuticalcomposition is an LBP polypeptide, e.g., LBP-1, LBP-2 or LBP-3, or abiologically active fragment or analog thereof. The agent can be, e.g.,the polypeptide as set forth in SEQ ID NOS:1-9. Preferably, the agent isa polypeptide of no more than about 100 amino acid residues in length,more preferably of no more than about 50 amino acid residues, morepreferably yet of no more than about 30 amino acid residues, morepreferably yet of no more than about 20 amino acid residues, morepreferably yet of no more than about 10 amino acid residues, morepreferably yet of no more than about 5 amino acid residues, morepreferably yet of no more than about 4 amino acid residues, morepreferably yet of no more than about 3 amino acid residues, and mostpreferably of no more than about 2 amino acid residues. Preferably, thepolypeptide includes at least about 20% acidic amino acid residues, morepreferably yet at least about 40% acidic amino acid residues, morepreferably yet at least about 60% acidic amino acid residues, morepreferably yet at least about 80% acidic amino acid residues, morepreferably yet at least about 90% acidic amino acid residues, morepreferably yet at least about 95% acidic amino acid residues, and mostpreferably at least about 98% acidic amino acid residues.

[0133] In certain preferred embodiments, the agent is an LBP nucleicacid, e.g., a nucleic acid encoding LBP-1, LBP-2 or LBP-3 polypeptide,or a biologically active fragment or analog thereof. The agent can be,e.g., a nucleic acid comprising a nucleotide sequence as set forth inSEQ ID NOS:10-18.

[0134] The invention also includes a vaccine composition for treatingatherosclerosis in an animal comprising a therapeutically effectiveamount of an agent, the agent being capable of altering an aspect of LBPmetabolism or structure in the animal so as to result in treatment ofthe atherosclerosis, and a pharmaceutically acceptable carrier.

[0135] The invention also includes a method for diagnosingatherosclerotic lesions in an animal. An animal is provided. A labeledagent capable of binding to LBP present in atherosclerotic lesions isprovided. The labeled agent is administered to the animal underconditions which allow the labeled agent to interact with the LBP so asto result in labeled LBP. The localization or quantification of thelabeled LBP is determined by imaging so as to diagnose the presence ofatherosclerotic lesions in the animal.

[0136] Preferably, the LBP is LBP-1, LBP-2 or LBP-3. The imaging can beperformed by standard methods known to those skilled in the art,including, e.g., magnetic resonance imaging, gamma camera imaging,single photon emission computed tomographic (SPECT) imaging, or positronemission tomography (PET).

[0137] Preferably, agents that bind tightly to LBPs in atheroscleroticlesions are used for atherosclerotic imaging and diagnosis. The agent isradiolabeled with, e.g., ^(99m)Tc or another isotope suitable forclinical imaging by gamma camera, SPECT, PET scanning or other similartechnology. Since LBPs occur in very early lesions, such imaging is moresensitive than angiography or ultrasound for locating very early lesionswhich do not yet impinge on the arterial lumen to cause a visible bulgeor disturbed flow. In addition to locating both early and more developedlesions, the imaging agents which bind to LBPs can also be used tofollow the progress of atherosclerosis, as a means of evaluating theeffectiveness of both dietary and pharmacological treatments.

[0138] Thus, a diagnostic embodiment of the invention is the adaptationof, e.g., a peptide complementary to one of the LBPs, by radiolabelingit and using it as an injectable imaging agent for detection of occultatherosclerosis. The peptide is selected from those known to bind toLBPs, e.g., RRRRRRR or KKLKLXX, or any other polycationic peptide whichbinds to the highly electronegative domains of the LBPs. Forextracorporeal detection with a gamma scintillation (Anger) camera,technetium-binding ligands, e.g., CGC, GGCGC, or GGCGCF, can beincorporated into the peptides at the N-terminus or C-terminus for^(99m)Tc labeling. For external imaging by magnetic resonance imaging(MRI), e.g., the gadolinium-binding chelator, diethylene triaminepenta-acetic acid (DTPA), is covalently bound to the N- or C-terminus ofthe peptides. In yet other embodiments, the LBP-binding peptides arecovalently bound, e.g., to magnetic ion oxide particles by standardmethods known to those skilled in the art, e.g., conjugating thepeptides with activated polystyrene resin beads containing magnetic ionoxide.

[0139] The invention also includes a method for immunizing an animalagainst an LBP, e.g., LBP-1, LBP-2 or LBP-3, or fragment or analogthereof. An animal having LDL is provided. An LBP or fragment or analogthereof is provided. The LBP or fragment or analog thereof isadministered to the animal so as to stimiulate antibody production bythe animal to the LBP or fragment or analog thereof such that binding ofthe LBP to the LDL is altered, e.g., decreased or increased.

[0140] The invention also includes a method of making a fragment oranalog of LBP polypeptide, the fragment or analog having the ability tobind to modified LDL and native LDL. An LBP polypeptide is provided. Thesequence of the LBP polypeptide is altered. The altered LBP polypeptideis tested for the ability to bind to modified LDL, e.g., methylated LDL,oxidized LDL, acetylated LDL, cyclohexanedione-treated LDL (CHD-LDL),and to native LDL.

[0141] The fragments or analogs can be generated and tested for theirability to bind to these modified LDLs and to native LDL, by methodsknown to those skilled in the art, e.g., as described herein.Preferably, they are tested for their ability to bind to methylated LDLand native LDL. The binding activity of the fragment or analog can begreater or less than the binding activity of the native LBP. Preferably,it is greater. In preferred embodiments, the LBP is LBP-1, LBP-2 orLBP-3.

[0142] The invention also includes a method for isolating a cDNAencoding an LBP. A cDNA library is provided. The cDNA library isscreened for a cDNA encoding a polypeptide which binds to native LDL andmodified LDL, e.g., methylated LDL or oxidized LDL. The cDNA whichencodes this polypeptide is isolated, the cDNA encoding an LBP.

[0143] The following non-limiting examples further illustrate thepresent invention.

EXAMPLES Example 1 Construction of a Rabbit cDNA Library

[0144] This example illustrates the construction of a rabbit cDNAlibrary using mRNA from balloon-deendothelialized healing rabbitabdominal aorta. Balloon-catheter deendothelialized rabbit aorta hasbeen shown to be a valid model for atherosclerosis (Minick et al., Am.J. Pathol. 95:131-158 (1979).

[0145] The mRNA was obtained four weeks after ballooning to maximizefocal LDL binding in the ballooned rabbit aorta. First strand cDNAsynthesis was carried out in a 50 μl reaction mixture containing 4 μgmRNA; 2 μg oligo d(T) primer; methylation dNTP mix (10 mM each); 10 mMDTT; 800 units superscript II RT (Life Technologies, Gaithersburg, MD);1× first strand cDNA synthesis buffer (50 mM Tris-HCl, pH 8.3; 75 mMKCl; 5 mM MgCl₂), which was incubated for 1 hr at 37° C. The reactionmixture was then adjusted to 250 μl through the addition of 1× secondstrand buffer (30 mM Tris-HCl, pH 7.5; 105 mM KCl; 5.2 mM MgCl₂); 0.1 mMDTT; methylation dNTP mix (10 mM each); 50 units E. coli DNA polymeraseI, 3 units RNase H; 15 units E. coli DNA ligase (all enzymes from LifeTechnologies), which was incubated for an additional 2.5 hr at 15° C.The resulting double-stranded cDNAs (dscDNA) were then treated with 1.5units T4 DNA polymerase (Novagen Inc., Madison, Wis.) for 20 min at 11°C. to make blunt-ended dscDNA. These were then concentrated by ethanolprecipitation and EcoR1/Hind III linkers were attached to the ends by T4DNA ligase (Novagen Inc.). The linker-ligated cDNAs were treated withEcoR1 and HindIII restriction enzymes to produce EcoR1 and Hind IIIrecognition sequences at their 5′ and 3′ ends, respectively. After theremoval of linker DNA by gel exclusion chromatography, the dscDNAs wereinserted into λEXlox phage arms (Novagen Inc.) in a unidirectionalmanner by T4 DNA ligase and packaged into phage particles according tothe manufacturer's protocol (Novagen Inc.). A phage library of cDNAscontaining 2×10⁶ independent clones was established from 4 μg of mRNA.

Example 2 Identification of Rabbit cDNAs Encoding LDL Binding Proteins(LBPs)

[0146] This example illustrates a method of functionally screening arabbit cDNA library so as to identify cDNAs encoding LBPs which bind toboth native LDL and methyl LDL. Methyl LDL is not recognized bypreviously reported cell surface receptors. See, e.g., Weisgraber etal., J. Biol. Chem. 253:9053-9062 (1978).

[0147] A fresh overnight culture of E. coli ER1647 cells (Novagen Inc.)was infected with the cDNA phage obtained from Example 1, and plated ata density of 2×10⁴ plaque-forming units (pfu) in 150 mm diameter platescontaining 2× YT agar. A total of 50 plates, equivalent to 1×10⁶ phage,were plated and incubated at 37° C. until the plaques reached 1 mm indiameter (5-6 hr). A dry nitrocellulose membrane, which had previouslybeen saturated with 10 mM IPTG solution, was layered on top of eachplate to induce the production of recombinant protein, as well as toimmobilize the proteins on the membranes. The plates were incubated at37° C. for an additional 3-4 hr, and then overnight at 4° C.

[0148] The next day, the membranes were lifted from each plate andprocessed as follows. Several brief rinses in TBST solution (10 mMTris-HCl, pH 8.0; 150 mM NaCl, 0.05% Tween 20); two 10-min rinses with6M guanidine-HCl in HBB (20 mM HEPES, pH 7.5; 5 mM MgCl₂, 1 mM DTT, and5 mM KCl); two 5-min rinses in 3M guanidine-HCl in HBB; a final briefrinse in TBSEN (TBS, 1 mM EDTA, 0.02% NaN₃).

[0149] The membranes were then blocked for 30 min at room temperature ina solution of TBSEN with 5% non-fat dry milk, followed by 10 min inTBSEN with 1% non-fat dry milk. Following blocking, the membranes wereincubated with native human LDL (obtained as described in Example 11 ormethylated human LDL (meLDL) (see Weisgraber et al., J. Biol. Chem.253:9053-9062 (1978)), at a concentration of 4 μg/ml, in a solutioncontaining 1× TBSEN, 1% non-fat dry milk, 1 mM PMSF, 0.5× proteaseinhibitor solution (1 mM ε-amino caproic acid/1 mM benzamidine).Incubation was for 4 hr at room temperature in a glass Petri dish withgentle stirring on a stirring table, followed by overnight at 4° C. withno stirring.

[0150] Specifically bound meLDL and native LDL were detected on thenitrocellulose membranes by antibodies against human LDL. Sheepanti-human LDL polyclonal antibodies (Boehringer Mannheim, Indianapolis,Ind.) were adsorbed with E. coli plys E cell extracts to abolishbackground. For adsorption, E. coli plys E cells were grown to logphase, spun down and resuspended in PBS containing 1 mM PMSF, 2 mMe-amino caproic acid, and 1 mM benzamidine. The cell suspension thenunderwent 8 freeze-thaw cycles via immersion in liquid nitrogen and coldrunning tap water, respectively. The anti LDL antibodies/cell extractsolution were incubated with gentle stirring for 1 hr at 4° C. (1 ml ofantibody solution/3 mg crude cell extract). Following incubation, themixture was centrifuged (10,000× g; 10 min; 4° C.) and the supernatantwas stored at 4° C. in the presence of 0.02% NaN₃ until use. Themembranes were processed for immunoscreening as follows: (i) three 5-minwashes at room temperature in TBSEN containing 1% gelatin; (ii) 30 minincubation in PBS, pH 7.4 with 1% gelatin; (iii) two-hr room temperatureincubation with gentle stirring in fresh PBS/gelatin solution containingadsorbed sheep anti-human LDL antibodies (Boehringer Manheim,Indianapolis, Ind.) (1:1000 dilution); (iv) three brief washes in TBS,pH 7.4; (v) one-hr room temperature incubation with gentle stirring inPBS/gelatin solution containing donkey antisheep alkalinephosphatase-conjugated antibodies (Sigma, St. Louis, Mo.) (1:10,000dilution); (vi) three brief washes with TBS, pH 7.4.; and (vii)development according to the manufacturer's instructions, using analkaline phosphatase substrate development kit (Novagen Inc.). Phageplaques which produced LBPs appeared as blue-colored “donuts” on themembranes.

[0151] The phage from Example 1 containing the LBP cDNAs wereplaque-purified and converted into plasmid subclones by following aprotocol called “Autosubcloning by Cre-mediated Plasmid Excision”provided by Novagen Inc. DNA sequences were obtained by thedideoxynucleotide chain-termination method (Sanger et al., Proc. Natl.Acad. Sci., USA 74:5463-5467 (1977), and analyzed by an AppliedBiosystems automated sequencer. The open reading frame (ORF) of eachcDNA was determined from consensus sequences obtained from both thesense and antisense strands of the cDNAs. Sequencing confirmed thatthree previously unknown genes had been isolated. Since the genes wereselected by functional screening for LDL binding, the proteins coded bythese genes were termed LDL binding proteins (LBPs), specifically,LBP-1, LBP-2 and LBP-3. The cDNA sequences for rabbit LBP-1, LBP-2 andLBP-3 and the corresponding proteins are set forth in SEQ ID NOS:10-14.

[0152] Based on their respective cDNA coding sequences, the sizes of therecombinant proteins were determined to be 16.2 kDa for LBP-1, 40 kDafor LBP-2, and 62.7 kDa for LBP-3.

Example 3 Northern Blot Analysis of Rabbit RNA Using LBP cDNA or cRNA

[0153] This example illustrates the size and tissue distribution of LBPmRNAs. Total RNA was isolated from different rabbit tissues: adrenals,thoracic aorta, abdominal aorta, ballooned and reendothelializedabdominal aorta, heart, kidney, smooth muscle cells, lung and liver, byTrizol reagent (Life Technologies) and concentrated by ethanolprecipitation. Gel electrophoresis of RNA was carried out in 1.2%agarose gel containing 1× MOPS buffer (0.2M MOPS, pH 7.0; 50 mM sodiumacetate; 5 mM EDTA, pH 8.0) and 0.37M formaldehyde. Gels were loadedwith 20 μg total RNA from each tissue examined and electrophoresed at100 volts for 2 hr in 1× MOPS buffer. RNAs were blotted onto supportednitrocellulose membranes (Schleicher & Schuell, Keene, N.H.) andimmobilized by baking at 80° C. for 2 hr. Hybridization to radiolabeledLBP-1, LBP-2 and LBP-3 cDNA or cRNA probes was carried out by standardprocedures known to those skilled in the art (see, e.g., Ausubel et al.,Current Protocols in Molecular Biology; John Wiley & Sons (1989));signals were detected by autoradiography.

[0154] The results were as follows: the sizes of the mRNAs were about1.3 kb for LBP-1, about 2.3-2.5 kb for LBP-2, and about 4.7 kb forLBP-3. LBP-1, LBP-2 and LBP-3 mRNA were found in all tissues tested, butthe highest amount was in ballooned abdominal aorta.

Example 4 Isolation of Human LBP cDNAs

[0155] This example illustrates isolation of human LBP cDNAs. Human LBPcDNA clones were isolated from three cDNA libraries. A human fetal braincDNA library was obtained from Stratagene, LaJolla, Calif., a humanliver and a human aorta cDNA library were obtained from Clontech, PaloAlto, Calif., and screened with a radiolabeled cDNA probe derived fromrabbit LBP-1, LBP-2 or LBP-3, according to the method described in Lawet al., Gene Expression 4:77-84 (1994). Several strongly hybridizingclones were identified and plaque-purified. Clones were confirmed to behuman LBP-1, LBP-2 and LBP-3, by DNA sequencing using thedideoxynucleotide chain-termination method and analysis by an AppliedBiosystems automated sequencer. The cDNA sequences and the correspondingproteins for human LBP-1, LBP-2 and LBP-3 are set forth in SEQ IDNOS:15, 16 and 17, respectively. A comparison between the correspondingLBP-1, LBP-2 and LBP-3 protein sequences for rabbit and human are shownin FIGS. 19, 20 and 21.

Example 5 Isolation of Recombinant LBP-1, LBP-2 and LBP-3 RabbitProteins from E. coli

[0156] LBP cDNA was isolated from the original pEXlox plasmids obtainedas described in Examples 1 and 2, and subcloned into the PPROEX-HTvector (Life Technologies) for recombinant protein expression. Inductionof the recombinant protein by IPTG addition to transformed E. coli DH10Bcultures resulted in the expression of recombinant protein containing a6-histidine tag (N-terminal). This tagged protein was then purified fromwhole cell proteins by binding to Ni-NTA (nickel nitrilo-triacetic acid)as described in the protocol provided by the manufacturer (Qiagen, Inc.,Santa Clara, Calif.). The preparation obtained after the chromatographystep was approximately 90% pure; preparative SDS-PAGE was performed asthe final purification step.

[0157] When required by the characterization procedure, iodination ofLBPs was carried out using Iodobeads (Pierce, Rockford, Ill.). TheIodobeads were incubated with 500 μCi of Na¹²⁵I solution (17 Ci/mg) (NewEngland Nuclear, Boston, Mass.) in a capped microfuge tube for 5 min atroom temperature. The protein solution was added to the Iodobeads-Na¹²⁵Imicrofuge tube and incubated for 15 min at room temperature. At the endof this incubation, aliquots were removed for the determination of totalsoluble and TCA precipitable counts. The radiolabeled protein was thenprecipitated with cold acetone (2.5 vol; −20° C.; 2.5 hr). Followingthis incubation, precipitated protein was collected by centrifugation(14,000 g; 1 hr; room temperature) and resuspended in sample buffer (6 Murea/50 mM Tris, pH 8.0/2 mM EDTA). Integrity of the protein preparationwas assessed by SDS-PAGE.

[0158] The identities of the recombinant LBPs were confirmed usingstandard protein sequencing protocols known to those skilled in the art.(A Practical Guide for Protein and Peptide Purification forMicrosequencing, Matsudaira, ed., Academic Press, Inc., 2d edition(1993)). Analysis was performed using an Applied Biosystems Model 477AProtein Sequencer with on-line Model 120 PTH amino acid analyzer.

Example 6 Production of Antibodies to LBP-1, LBP-2 and LBP-3

[0159] This example illustrates the production of polyclonal antibodiesto LBP-1, LBP-2 and LBP-3. A mixture of purified recombinant LBP protein(0.5 ml; 200 μg) and RIBI adjuvant (RIBI ImmunoChem Research, Inc.,Hamilton, Mont.) was injected subcutaneously into male guinea pigs(Dunkin Hartley; Hazelton Research Products, Inc., Denver, Pa.) at 3-5sites along the dorsal thoracic and abdominal regions of the guinea pig.Blood was collected by venipuncture on days 1 (pre-immune bleeding), 28,49 and 70. Booster injections were administered on days 21 (100 μg; SC),42 (50 μg; SC), and 63 (25 μg; SC). The titer of the guinea pigantiserum was evaluated by serial dilution “dot blotting.” Preimmuneantiserum was evaluated at the same time. After the third booster of LBPprotein, the titer against the recombinant protein reached a maximallevel with a detectable calorimetric response on a dot blot assay of 156pg.

[0160] Specificity of the polyclonal antibody for recombinant LBP-1,LBP-2 or LBP-3 was demonstrated using Western blot analysis. (Towbin etal., Proc. Natl. Acad. Sci. USA 76:4350 (1979)). The protein-antibodycomplex was visualized immunochemically with alkalinephosphatase-conjugated goat anti-guinea pig IgG, followed by stainingwith nitro blue tetrazolium (BioRad Laboratories, Hercules, Calif.).Non-specific binding was blocked using 3% non-fat dry milk in Trisbuffered saline (100 mM Tris; 0.9% NaCl, pH 7.4).

Example 7 Immunohistochemical Characterization

[0161] This example illustrates the presence of LBPs in or onendothelial cells covering plaques, in or on adjacent smooth musclecells, and in the extracellular matrix. In addition, co-localization ofLDL and LBPs was demonostrated. These results were obtained by examiningballooned rabbit arterial lesions and human atherosclerotic plaques byimmunohistochemical methods.

[0162] Ballooned deendothelialized aorta was obtained from rabbits whichhad received a bolus injection of human LDL (3 mg; i.v.) 24 hr prior totissue collection. Human aortas containing atherosclerotic plaques wereobtained from routine autopsy specimens. Tissues were fixed in 10%buffered formalin (≦24 hr) and imbedded in paraffin using an automatedtissue-imbedding machine. Tissue sections were cut (5-7μ) and mountedonto glass slides by incubating for 1 hr at 60° C. Sections weredeparaffinized. After a final wash with deionized H₂O, endogenousperoxidase activity was eliminated by incubating the sections with 1%H₂O₂/H₂O buffer for 5 min at room temperature. Sections were rinsed withphosphate buffered saline (PBS) for 5 min at room temperature andnonspecific binding was blocked with 5% normal goat serum or 5% normalrabbit serum depending on the source of the secondary antibody (Sigma,St. Louis, Mo.) (1 hr; room temperature). Sections were then incubatedwith a 1:50 dilution (in 5% normal goat serum/PBS) of a guinea pigpolyclonal antibody against the rabbit form of recombinant LBP-1, LBP-2or LBP-3. Controls included preimmune serum as well as specific antiserato LBP-1, LBP-2, or LBP-3 in which the primary antibody was completelyadsorbed and removed by incubation with recombinant LBP-1, LBP-2 orLBP-3 followed by centrifugation prior to incubation with the tissuesections. An affinity purified rabbit polyclonal antibody against humanapolipoprotein B (Polysciences Inc.; Warrington, Pa.) was used at adilution of 1:100 (in 5% normal rabbit serum/PBS). Sections wereincubated for 2 hr at room temperature in a humidified chamber. At theend of incubation, sections were rinsed with PBS and incubated with a1:200 dilution (in 5% normal goat serum/PBS) of goat anti-guinea pigbiotinylated IgG conjugate (Vector Laboratories, Burlingame, Calif.) ora 1:250 dilution (in 5% normal rabbit serum/PBS) of rabbit anti-goatbiotinylated IgG conjugate (Vector Laboratories, Burlingame, Calif.) for1 hr at room temperature in a humidified chamber. Sections were thenrinsed with PBS and antigen-antibody signal amplified usingavidin/biotin HRP conjugate (Vectastain ABC kit; Vector Laboratories,Burlingame, Calif.). Sections were developed using DAB substrate (4-6min; room temperature) and counterstained with hematoxylin.

[0163] In the ballooned rabbit artery, immunohistochemistry with theanti-LBP-1, LBP-2 and LBP-3 antibodies showed that LBP-1, LBP-2 andLBP-3 were located in or on functionally modified endothelial cells atthe edges of regenerating endothelial islands, the same location inwhich irreversible LDL binding has been demonstrated (Chang et al.,Arteriosclerosis and Thrombosis 12:1088-1098 (1992)). LBP-1, LBP-2 andLBP-3 were also found in or on intimal smooth muscle cells underneaththe functionally modified endothelial cells, and to a lesser extent, inextracellular matrix. No LBP-1, LBP-2 or LBP-3 was detected in stilldeendothelialized areas, where LDL binding had been shown to bereversible (Chang et al., Arteriosclerosis and Thrombosis 12:1088-1098(1992)). Immunohistochemistry of ballooned rabbit aorta with anti-humanapolipoprotein B antibodies showed the presence of LDL at the samelocations as that found for LBP-1, LBP-2 and LBP-3.

[0164] In the human atherosclerotic plaques taken at routine autopsies,immunohistochemistry with the anti-LBP-1, anti-LBP-2 and anti-LBP-3antibodies showed that LBP-1, LBP-2, and LBP-3 were also found in or onendothelial cells covering plaques and in or on adjacent smooth musclecells. In the human tissue, there was greater evidence of LBP-1, LBP-2and LBP-3 in extracellular matrix.

[0165] The results obtained with paraffin sections were identical tothose of frozen sections.

Example 8 Affinity Coelectrophoresis (ACE) Assays of LBPs and LDL or HDL

[0166] This example illustrates that binding occurs between LBP-1, LBP-2or LBP-3 and LDL, and that this binding is specific, as illustrated bythe fact that binding does not occur between LBP-1, LBP-2 or LBP-3 andHDL (high density lipoprotein).

[0167] Analysis of the affinity and specificity of recombinant rabbitLBP-1, LBP-2 or LBP-3 binding to LDL was carried out using the principleof affinity electrophoresis (Lee and Lander, Proc. Natl. Acad. Sci. USA88:2768-2772 (1991)). Melted agarose (1%; 65° C.) was prepared in 50 mMsodium MOPS, pH 7.0; 125 mM sodium acetate, 0.5% CHAPS. A teflon combconsisting of nine parallel bars (45×4×4 mm/3 mm spacing between bars)was placed onto GelBond film (FMC Bioproducts, Rockland, Me.) fitted toa plexiglass casting tray with the long axis of the bars parallel to thelong axis of the casting tray. A teflon strip (66×1×1 mm) was placed onedge with the long axis parallel to the short axis of the casting tray,at a distance of 4 mm from the edge of the teflon comb. Melted agarose(>65° C.) was then poured to achieve a height of approximately 4 mm.Removal of the comb and strip resulted in a gel containing nine 45×4×4mm rectangular wells adjacent to a 66×1 mm slot. LDL or HDL samples wereprepared in gel buffer (50 mM sodium MOPS, pH 7.0, 125 mM sodiumacetate) at twice the desired concentration. Samples were then mixedwith an equal volume of melted agarose (in 50 mM MOPS, pH 7.0; 125 mMsodium acetate; 50° C.), pipetted into the appropriate rectangular wellsand allowed to gel. The binding affinity and specificity of LBP-1 andLBP-3 was tested using several concentrations of LDL (540 to 14 nM) andHDL (2840-177 nM). A constant amount (0.003 nM-0.016 nM) of ¹²⁵I-labeledLBP-1, LBP-2 or LBP-3 (suspended in 50 mM sodium MOPS, pH 7.0; 125 mMsodium acetate; 0.5% bromphenol blue; 6% (wt/vol) sucrose) was loadedinto the slot. Gels were electrophoresed at 70 v/2 hr/20° C. At the endof the run, the gels were air dried and retardation profiles werevisualized by exposure of X-ray films to the gels overnight at −70° C.,with intensifying screens).

[0168] LDL retarded LBP-1, LBP-2 and LBP-3 migration through the gel ina concentration-dependent, saturable manner, indicating that LBP-1,LBP-2 and LBP-3 binding to LDL was highly specific. This conclusion issupported by the fact that HDL did not retard LBP-1, LBP-2 or LBP-3. Abinding curve generated from the affinity coelectrophoresis assayindicated that LBP-1 binds to LDL with a K_(d) of 25.6 nM, that LBP-2(rabbit clone 26) binds to LDL with a K_(d) of 100 nM, and that LBP-3(80 kDa fragment) binds to LDL with a K_(d) of 333 nM.

[0169] In addition to testing affinity and specificity of LBP-1, LBP-2and LBP-3 binding to LDL, the ability of “cold” (i.e., non-radiolabeled)LBP-1, LBP-2 or LBP-3 to competitively inhibit radiolabeled LBP-1, LBP-2or LBP-3 binding to LDL, respectively, was tested. Competition studieswere carried out using fixed concentrations of cold LDL and radiolabeledLBP-1 and increasing amounts of cold recombinant LBP-1 (6-31 μM). TheACE assay samples and gel were prepared as described herein. Cold LBP-1inhibited binding of radiolabeled LBP-1 to LDL in aconcentration-dependent manner, cold LBP-2 inhibited binding ofradiolabeled LBP-2 to LDL in a concentration-dependent manner, and coldLBP-3 inhibited binding of radiolabeled LBP-3 to LDL in aconcentration-dependent manner.

[0170] Rabbit and human LBP-2 contain a long stretch of acidic aminoacids at the amino terminal (rabbit LBP-2 amino acid residues 105through 132 and human LBP-2 amino acid residues 8 through 33). Thepossibility that this segment of LBP-2 was the LDL binding domain wastested by subcloning two rabbit LBP-2 clones which differ from eachother by the presence or absence of this acidic region (clone 26 andclone 45, respectively) into expression vectors, by standard methodsknown to those skilled in the art. ACE assays were then conducted inorder to assess the affinity and specificity of the binding of these twoclones to LDL. LDL retarded clone 26 derived radiolabeled LBP-2migration through the gel in a concentration-dependent, saturable,manner while clone 45 derived radiolabeled LBP-2 migration was notretarded.

[0171] Competition studies using fixed concentrations of cold LDL andclone 26 derived radiolabeled LBP-2 and increasing concentrations ofcold recombinant LBP-2/clone 26 and LBP-2/clone 45 were carried out.Cold clone 26 derived LBP-2 inhibited binding of clone 26 derivedradiolabeled LBP-2 to LDL in a concentration-dependent manner. Clone 45derived LBP-2, on the other hand, did not affect the binding of clone 26derived radiolabeled LBP-2 to LDL. These results indicate that the longstretch of acidic amino acids contain a binding domain of LBP-2 to LDL.

Example 9 Affinity Coelectrophoresis (ACE) Assays of LBP-1 or LBP-2 andLDL in the Presence of Inhibitors

[0172] This example illustrates that binding between LBP-1 or LBP-2 andLDL is inhibited by polyglutamic acid or BHF-1. The ability of a thirdcompound to inhibit binding between two proteins previously shown tointeract was tested by a modification of the ACE assays described inExample 8. The third compound was added to the top or wells togetherwith the radiolabeled protein. If the third compound inhibited binding,the radiolabeled protein would run through the gel. If the thirdcompound did not inhibit binding, migration of the radiolabeled proteinwas retarded by the protein cast into the gel.

[0173] Inhibition of LBP-l/LDL or LBP-2/LDL binding by polyglutamic acid(average MW about 7500, corresponding to about 7 monomers) was shown bycasting a constant amount of LDL (148 nM) in all the rectangular lanes.A constant amount (1 μl) of ¹²⁵I-labeled LBP-1 or LBP-2 (0.003 nM-0.016nM) was loaded in the wells at the top of the gel, together withincreasing concentrations of polyglutamic acid (obtained from Sigma)(0-0.4 nM). The gel was electrophoresed at 70 volts for 2 hr, dried andplaced on X-ray film, with intensifying screens, overnight at −70° C.before the film was developed to determine the retardation profile ofLBP-1 and LBP-2. As the concentration of polyglutamic acid increased,retardation of radiolabeled LBP-1 and LBP-2 migration by LDL decreasedin a concentration-dependent manner, which showed that polyglutamic acidinhibited binding between LBP-1, LBP-2 and LDL.

[0174] Inhibition of LBP-1/LDL binding by BHF-1 was shown by casting aconstant amount of LDL (148 nM) in all the rectangular lanes. A constantamount of ¹²⁵I-labeled LBP-1 (0.003 nM-0.016 nM) was loaded in the wellsat the top of the gel, together with increasing concentrations of BHF-1(0-10 nM), obtained as described in Example 15. The gel waselectrophoresed at 70 volts for 2 hr, dried and placed on X-ray film,with intensifying screens, overnight at −70° C. The film was thendeveloped to determine the retardation profile of ¹²⁵I-LBP-1. As theconcentration of BHF-i increased, retardation of LBP-1 by LDL decreasedin a concentration-dependent manner, which demonstrated that BHF-1inhibited binding between LBP-1 and LDL.

Example 10 Affinity Coelectrophoresis (ACE) Assays for IdentifyingFragments, Analogs and Mimetics of LBPs which Bind to LDL

[0175] This example illustrates a method for identifying fragments,analogs or mimetics of LBPs which bind to LDL, and which thus can beused as inhibitors of LDL binding to LBP in the arterial walls, byoccupying binding sites on LDL molecules, thereby rendering these sitesunavailable for binding to LBP in the arterial wall.

[0176] Fragments of LBPs are generated by chemical cleavage orsynthesized from the known amino acid sequences. Samples of thesefragments are individually added (cold) to radiolabeled LBP as describedin Example 8, to assess the inhibitory potency of the various fragments.By iterative application of this procedure on progressively smallerportions of fragments identified as inhibitory, the smallest activepolypeptide fragment or fragments are identified. In a similar manner,analogs of the LBPs are tested to identify analogs which can act asinhibitors by binding to LDL. And, similarly. mimetics of LBP (moleculeswhich resemble the conformation and/or charge distributions of theLDL-binding sites on LBP molecules) are tested in a similar fashion toidentify molecules exhibiting affinities for the LDL-binding sites onLBP.

[0177] The affinities of the inhibitors so identified are at least asstrong as the affinity of LDL itself for the LDL-binding sites on LBP.The inhibitors bind at least competitively, and some irreversibly andpreferentially as well, to the LDL-binding sites, thereby rendering suchsites unavailable for binding to humoral LDL.

Example 11 ELISA Assays

[0178] This example illustrates the use of an ELISA plate assay for thequantification of a test compound's capacity to inhibit the binding ofLDL to a specific LBP.

[0179] The assay was carried out as follows: LDL was diluted in 50 mMNa₂HCO₃, pH 9.6/0.02% NaN₃ and added to the wells of a 96-well plate(ImmunoWare 96-Well Reacti-Bind EIA Polystyrene Plates; Pierce(Rockford, Ill.)) to achieve a final concentration ranging from 0.1 to 1μg/well. The plates were incubated for 6 hr at room temperature. At theend of the incubation period, the wells were washed 3 times withTris-buffered saline, pH 7.4 (TBS), and blocked overnight with 200 μl of1% bovine serum albumin (BSA) in TBS/0.02% NaN₃ (Sigma; St. Louis Mo.)at room temperature. The wells were then incubated with 200 μl of LBPprotein (5-10 μg/well) in TBS and varying concentrations of the testcompound. Plates were incubated for 1 hr at room temperature. The wellswere then washed three times with TBS and blocked for 2 hr with 200 μlof 1% BSA in TBS/0.02% NaN₃ at room temperature. At the end of theincubation period, the wells were washed 3 times with TBS and a 1:1000dilution (in TBS/0.05% Tween 20) of the appropriate guinea pig anti-LBPprotein polyclonal antibody was added to the wells and incubated for 1hr at room temperature. The wells were then washed 3 times withTBS/0.05% Tween 20; a 1:30,000 dilution of goat anti-guinea pig IgGalkaline phophatase conjugate (Sigma) was added to each well. Plateswere incubated for 1 hr at room temperature. The wells were washed 3times with TBS/0.05% Tween 20 and a calorimetric reaction was carriedout by adding 200 ml of p-nitrophenyl phosphate substrate (Sigma; St.Louis Mo.) to the wells. The reaction was allowed to proceed for 30 minat room temperature and stopped with 50 μl of 3N NaOH. The absorbancewas determined at 405 nm using an ELISA plate reader. The testcompound's effectiveness in blocking the binding of LDL to therecombinant protein was assessed by comparing the absorbance values ofcontrol and treated groups.

[0180] Alternatively, LBPs, rather than LDL, were bound to the plate.Recombinant LBP protein binding to LDL and the effect of varyingconcentration of the inhibitor on LBP-LDL binding was determined throughthe use of antibodies against LDL. This interaction was visualizedthrough the use of a secondary antibody conjugated to a reporter enzyme(e.g. alkaline phosphatase).

[0181] ELISA plate assays were used to screen agents which can affectthe binding of LBP proteins to LDL. For example, peptides derived fromLBP-1 and human LBP-3 protein sequences (BHF-1 and BHF-2, respectively)were synthesized and have been shown to reduce the binding of LDL torecombinant LBP-1 and LBP-2 in this format. These results were inagreement with those obtained with the ACE assays.

Example 12 Administration of Humanized Antibodies Against LBPs so as toBlock LDL-binding Sites on the LBPs

[0182] This example illustrates administration to patients of humanizedantibodies against LBP-1, LBP-2 or LBP-3 so as to block LDL-bindingsites on arterial LBP molecules. Mouse monoclonal antibodies arehumanized by recombinant DNA techniques and produced by standardprocedures known to those skilled in the art (Berkower, I., Curr. Opin.Biotechnol. 7:622-628 (1996); Ramharayan and Skaletsky, Am. Biotechnol.Lab 13:26-28 (1995)) against LBPs and/or the LDL-binding sites on theLBPs. The corresponding Fab fragments are also produced, as described inGoding, J. W., Monoclonal Antibodies:Principles and Practice, AcademicPress, New York, N.Y. (1986). These antibodies are administeredparenterally in sufficient quantity so as to block LDL-binding sites onthe LBP molecules, i.e., 1-10 mg/kg daily. This prevents theirreversible arterial uptake of LDL that is required to facilitateoxidation of the LDL.

Example 13 Preparation of LDL

[0183] This example illustrates the preparation of LDL. LDL was preparedfrom the plasma of normolipemic donors (Chang et al., Arterioscler.Thromb. 12:1088-1098 (1992)). 100 ml of whole blood was placed intotubes containing 100 mM disodium EDTA. Plasma was separated from redblood cells by low-speed centrifugation (2,000 g; 30 min; 4° C.). Plasmadensity was adjusted to 1.025 gm/ml with a solution of KBr andcentrifuged for 18-20 hr, 100,000× g, 12° C. Very low densitylipoproteins (VLDL) were removed from the tops of the centrifuge tubeswith a Pasteur pipet. The density of the infranate was raised to 1.050gm/ml with KBr solution and centrifuged for 22-24 hr, 100,000× g, 12° C.LDL was removed from the tops of the centrifuge tubes with a drawn outPasteur pipet tip. Purity of the LDL preparation was checked byOuchterlony double immunodiffusion against antibodies to human LDL,human HDL, human immunoglobulins, and human albumin. KBr was removedfrom the LDL solution by dialysis (1 L,×2,≈16 hr) against 0.9% saline,pH 9.0, containing 1 mM EDTA and 10 μM butylated hydroxytoluene (BHT),the latter to prevent oxidation of LDL. Following dialysis, LDL proteinwas measured by the method of Lowry (Lowry et al., J. Biol. Chem.193:265-275 (1951)), and the LDL was stored at 4° C. until use. LDLpreparations were kept for no more than 4-6 weeks.

Example 14 Preparation of HDL

[0184] This example illustrates the preparation of HDL. HDL was preparedfrom plasma of normolipemic donors. 100 ml of whole blood was placedinto tubes containing 100 mM disodium EDTA and plasma was collected bycentrifugation (2000 g; 30 min; 4° C.). Apolipoprotein B containinglipropoteins present in plasma were then precipitated by the sequentialaddition of sodium heparin (5,000 units/ml) and MnCl₂ (1M) to achieve afinal concentration of 200 units/ml and 0.46 M, respectively (Warnickand Albers, J. Lipid Res. 19:65-76 (1978)). Samples were thencentrifuged (2000 g; 1 hr; 4° C.). The supernatant was collected anddensity adjusted to 1.21 g/ml by the slow addition of solid KBr. HDL wasseparated by ultracentrifugation (100,000 g; >46 hr; 12° C.). Purity ofthe HDL preparation was assessed via Ouchterlony double immunodiffusiontest using antibodies against human HDL, human LDL, humanimmunoglobulins, and human albumin. HDL samples were dialyzed againstsaline pH 9.0/1 mM EDTA/10 μM BHT (4 L; 24 hr/4° C.) and total proteinwas determined by the Lowry protein assay (Lowry et al., J. Biol. Chem.193:265-275 (1951)). HDL was stored at 4° C. until use. HDL preparationswere kept for no longer than 2 weeks.

Example 15 Synthesis of BHF-1

[0185] This example illustrates the synthesis of BHF-1, a fragment ofhuman or rabbit LBP-1 which contains amino acid residues 14 through 33.BHF-1 was synthesized using an Applied Biosystems Model 430A peptidesynthesizer with standard T-Boc NMP chemistry cycles. The sequence ofBHF-1 is as follows:

[0186]val-asp-val-asp-glu-tyr-asp-glu-asn-lys-phe-val-asp-glu-glu-asp-gly-gly-asp-gly(SEQ ID NO:9)

[0187] After synthesis, the peptide was cleaved with hydrofluoricacid/anisole (10/1 v/v) for 30 min at −10° C. and then incubated for 30min at 0° C. BHF-1 was then precipitated and washed three times withcold diethyl ether. Amino acid coupling was monitored with the ninhydrintest (>99%).

[0188] The BHF-1 peptide was purified to homogeneity by high performanceliquid chromatography on a reverse phase Vydac C4 column (2.24×25 cm)using a linear gradient separation (2-98% B in 60 min) with a flow rateof 9 ml/min. Buffer A consisted of 0.1% trifluoroacetic acid (TFA)/MilliQ water and Buffer B consisted of 0.085% TFA/80% acetonitrile. Thegradient was run at room temperature and absorbance monitored at 210 and277 nm.

[0189] Fast atom bombardment-mass spectrometry gave a protonatedmolecular ion peak (M+H)⁺ at m/z=2290.2, in good agreement with thecalculated value. On amino acid analysis, experimental values for therelative abundance of each amino acid in the peptide were in goodagreement with theoretical values. The lyophilized peptide was stored at−20° C.

Example 16 In vitro Screening for Agents Which Inhibit Binding BetweenLDL and LBPs

[0190] This example illustrates in vitro screening for agents whichinhibit binding between LDL and LBPs.

[0191] A candidate polypeptide for being an agent is chosen, e.g.,LBP-1, LBP-2, LBP-3, BHF-1 or any other polypeptide. The shortestfragment of the polypeptide that inhibits LDL binding to LBPs in vitrois determined. Peptides are synthesized by standard techniques describedherein. Inhibition assays are performed using standard ELISA techniquesfor screening, and affinity coelectrophoresis (ACE) assays to confirmthe ELISA results, as described herein. Short peptides ranging, e.g.,from dimers to 20-mers are constructed across sequences of the candidatepolypeptide whose chemical characteristics make them likely LDL bindingsites, e.g., acidic regions. The ability of shorter and shorter lengthsof the peptides to inhibit LDL binding in vitro and to mammalian cellsin culture is tested. For example, the effect of the peptide oninhibiting LDL binding in mammalian cells transfected to express an LBPgene is tested. Each of the peptides so identified as an inhibitor istested with each of LBP-1, LBP-2 and LBP-3, to determine whether asingle inhibitor works against all three LBPs.

[0192] Once the minimum active sequence is determined, the peptidebackbone is modified so as to inhibit proteolysis, as discussed herein.For example, modification is accomplished by substitution of a sulfoxidefor the carbonyl, by reversing the peptide bond, by substituting amethylene for the carbonyl group, or other similar standard methodology.See Spatola, A. F., “Peptide Backbone Modifications: AStructure-Activity Analysis of Peptides Containing Amide BondSurrogates, Conformational Constraints, and Related BackboneReplacements,” in Chemistry and Biochemistry of Amino Acids, Peptidesand Proteins, Vol. 7, pp. 267-357, B. Weinstein (ed.), Marcel Dekker,Inc., New York (1983). The ability of these analogs to inhibit LDLbinding to the LBPs in vitro is tested by ELISA and ACE assays in asimilar manner as for the natural peptides described above.

Example 17 In vitro Screening With Cultured Mammalian Cells for AgentsWhich Inhibit Binding Between LDL and LPBs

[0193] This example illustrates cell-based in vitro screening of agentswhich have been shown by in vitro tests such as ACE assay and ELISA tobe potential inhibitors of binding between LDL and LBPs.

[0194] Mammalian cells, such as 293 cells, which are commonly used forexpression of recombinant gene constructs, are used to develop celllines which express LBPs on the cell surface. This is done by subcloningLBP open reading frames (ORFs) into a mammalian expression plasmidvector, pDisplay (Invitrogen, Carlsbad, Calif.), which is designed toexpress the gene of interest on the cell surface. The use of mammaliancells to produce LBPs allows for their expression in a functionallyactive, native conformation. Therefore, stably transfected mammaliancell lines with surface expression of LBPs individually, or incombination, are particularly suitable for assaying and screeninginhibitors that block LDL binding in cell culture, as well as toevaluate the cytotoxicity of these compounds.

[0195] Specifically, LBP ORFs are amplified by PCR (Perkin Elmer, FosterCity, Calif.) from cDNA templates using Taq polymerase (Perkin Elmer)and appropriate primers. The amplified LBP ORFs are purified by agarosegel electrophoresis and extracted from gel slices with the Bio-Rad DNAPurification kit (Bio-Rad, Hercules, Calif.). The purified DNAs are thencut with the restriction enzymes Bgl II and Sal I (New England Biolabs,Beverly, Mass.) to generate cohesive ends, and purified again by agarosegel electrophoresis and DNA extraction as described above. The LBP ORFsare then subcloned into the Bgl II/Sal I sites in the mammalianexpression vector, pDisplay (Invitrogen) by ligation. Recombinantplasmids are established by transformation in E.coli strains TOP10(Invitrogen) or DH5α (Life Technologies, Grand Island, N.Y.).Recombinant pDisplay/LBP plasmid DNA is isolated from overnight E.colicultures with the Bio-Rad Plasmid Miniprep kit, cut with Bgl II/Sal I,and analyzed by agarose gel electrophoresis. LBP ORFs in successfullytransformed clones are verified by automated dideoxy DNA sequencing. Totransfect human kidney 293 cells, 1-2 μg of DNA is mixed with 6 μlliofectamine reagent (Life Technologies) and incubated with the cells asdescribed in the Life Technologies protocol. LBP expression intransfected cells is confirmed by Western blot analysis of cell extractsobtained 48 hr after transfection. To select for stably transfected 293cells, the antibiotic G418 (Life Technologies) is added to the growthmedium at a concentration of 800 μg/ml. Colonies resistant to G418 aretested for recombinant LBP expression by Western blot, and recombinantclones expressing LBPs are expanded, assayed for LDL binding and used totest compounds for their ability to inhibit LDL binding.

Example 18 In vivo Screening for Agents Which Inhibit Binding BetweenLDL and LBPs

[0196] This example illustrates in vivo screening of agents which havebeen shown by in vitro tests to be promising candidate inhibitors ofbinding between LDL and LBPs.

[0197] In vivo inhibitory activity is first tested in the healingballoon-catheter deendothelialized rabbit aorta model of arterial injury(Roberts et al., J. Lipid Res. 24:1160-1167 (1983); Chang et al.,Arterioscler. Thomb. 12:1088-1098 (1992)). This model was shown to be anexcellent analog for human atherosclerotic lesions. Each candidateinhibitor is tested in five to ten ballooned rabbits, while an equalnumber of rabbits receive a control peptide, or placebo. Four weeksfollowing aortic deendothelialization, when reendothelialization(healing) is partially complete, daily parenteral (intravenous orsubcutaneous) or intragastric administration of the peptides and theanalogs begins at an initial concentration of 10 mg/kg body weight,which is varied down, or up to 100 mg/kg depending on results. 30 minlater, a bolus of intravenously injected ¹²⁵I (or ^(99m)Tc-) labeled LDLis given to zest the candidate inhibitor's ability in short term studiesto inhibit LDL sequestration in healing arterial lesions. If ¹²⁵I-LDL isused, the animals are sacrificed 8-24 hr later, the aortas excised,washed and subjected to quantitative autoradiography of excised aortas,as previously described (Roberts et al., J. Lipid Res. 24:1160-1167(1983); Chang et al., Arterioscler. Thomb. 12:1088-1098 (1992)). If^(99m)Tc-LDL is used, analysis is by external gamma camera imaging ofthe live anesthetized animal at 2-24 hr, as previously described (Leesand Lees, Syndromes of Atherosclerosis, in Fuster, ed., FuturaPublishing Co., Armonk, N.Y., pp. 385-401 (1996)), followed bysacrifice, excision and imaging of the excised aorta. Immediately beforethe end of testing, the animals have standard toxicity tests, includingCBC, liver enzymes, and urinalysis.

[0198] The compounds which are most effective and least toxic are thentested in short term studies of rabbits fed a 2% cholesterol diet(Schwenke and Carew, Arteriosclerosis 9:895-907 (1989)). Each candidateinhibitor is tested in five to ten rabbits, while an equal number ofrabbits receive a control peptide, or placebo. Animals receive one ormore doses per day of the candidate inhibitor, or placebo, for up to twoweeks. Daily frequency of doses is determined by route ofadministration. If active drug or placebo are administered parenterally,they are given 1-3 times daily and the 2% cholesterol diet is continued.If drug or placebo are given orally, they are mixed with the 2%cholesterol diet. Schwenke and Carew (Arteriosclerosis 9:895-907 (1989))have shown that the LDL concentration in lesion-prone areas of therabbit aorta is increased 22-fold above normal in rabbits fed a 2%cholesterol diet for 16 days, and that the increased LDL contentprecedes the histological evidence of early atherosclerosis. Therefore,analysis of the effect of the candidate inhibitors is tested two weeksafter the start of cholesterol feeding by injecting ¹²⁵I-LDL, allowingit to circulate for 8-24 hr, and then performing quantitativeautoradiography on the excised aortas of both test and control animals.If appropriate, quantitation of aortic cholesterol content is alsocarried out (Schwenke and Carew, Arteriosclerosis 9:895-907 (1989);Schwenke and Carew, Arteriosclerosis 9:908-918 (1989).

[0199] The above procedures identify the most promising candidateinhibitors, as well as the best route and frequency of theiradministration. Inhibitors so identified are then tested in long-termstudies of cholesterol-fed rabbits. These tests are carried out in thesame way as the short-term cholesterol feeding studies, except thatinhibitor effectiveness is tested by injection of ¹²⁵I-LDL at longerintervals following the initiation of cholesterol feeding, andlesion-prone areas of the aorta are examined histologically for evidenceof atherosclerosis. Testing times are at two, four, and six months.Major arteries are examined grossly and histologically for evidence andextent of atherosclerosis. If necessary, other accepted animal models,such as atherosclerosis-susceptible primates (Williams et al.,Arterioscler. Thromb. Vasc. Biol. 15:827-836 (1995) and/or Watanaberabbits are tested with short- and long-term cholesterol feeding.

Example 19 In vivo Inhibition of Radiolabeled LDL Accumulation in theBallooned Deendothelialized Rabbit Aorta via Induction of ActiveImmunity Against LBP Protein

[0200] This example illustrates the effect that induction of immunityagainst LBP protein has on the accumulation of radiolabeled LDL in theballooned deendothelialized rabbit aorta model of atherosclerosis.

[0201] Immunity was induced in male New Zealand White rabbits (HazeltonResearch Products, Denver, Pa.) as follows: A mixture of purified humanrecombinant LBP-2 or BHF-1 peptide (1 ml; 1 mg) and RIBI adjuvant (RIBIImmunoChem Research, Inc., Hamilton, Mont.) was injected subcutanouslyat 2-5 sites along the dorsal thoracic and abdominal regions of therabbits. Blood was collected by venipuncture on days 1 (preimmunebleeding), 35, 63, and 91. Booster injections were administered on days28 (500 μg; SC), 56 (250 μg; SC), and 84 (125 μg; SC).

[0202] The titer of the rabbits was evaluated by serial dilution usingan ELISA plate format. Preimmune serum was evaluated at the same time.After the third booster of LBP protein or peptide, the titer reached amaximal level with a detectable calorimetric response on an ELISA plateof 156 pg. Titer is defined as the maximum dilution of antibody whichgenerates an absorbance reading of 0.5 above control in 30 min.Specificity of the polyclonal antibodies was demonstrated using Westernblot analysis as described in Example 6.

[0203] On day 93, the abdominal aorta of immunized and control rabbitswas deendothelialized using a Fogarty number 4 embolectomy catheter(Chang et al., Arteriosclerosis and Thrombosis 12:1088-1098 (1992)).Four weeks after ballooning, rabbits received a bolus injection of¹²⁵I-labeled LDL (1 ml; i.v.). Blood samples were collected at 1 hrintervals for 8 hr, and 24 hr post injection. Blood samples werecentrifuged for 30 min at 2000 rpm (40° C.) and total activity presentin the serum was determined using a Gamma counter. Total TCAprecipitable counts were determinined by addition of TCA to the serum toa final concentration of 10% followed by incubation for 10 min at 4° C.Serum samples were then centrifuged (2000 rpm; 30 min; 40° C.) and totalactivity present in the supernate was determined. TCA precipitablecounts were calculated by substration: total soluble counts minus countspresent in the supernate after TCA precipitation. Blood samples for thedetermination of antibody titers were collected prior to the injectionof the radiolabeled LDL.

[0204] After 24 hr, the rabbits were injected intravenously with 5%Evan's blue dye which was allowed to circulate for 15 min. Areas of theaorta in which the endothelial covering is absent stain blue while thoseareas covered by endothelium remain unstained. At the end of theincubation period, the rabbits were euthanized and the abdominal andthoracic aorta were dissected out, rinsed, and fixed overnight in 10%TCA at room temperature. The aortas were then rinsed exhaustively withphysiological saline, weighed, counted, blotted dry and placed ontoX-ray film in order to visualize the pattern of radiolabeled LDLaccumulation in the deendothelialized rabbit abdominal aorta.

[0205] Immunization of rabbits against recombinant human LBP-2 or BHF-1peptide altered the pattern of radiolabeled LDL accumulation in theballooned deendothelialized abdominal aorta. When corrected for dosage,and percent reendothelialization, immunized-ballooned rabbits had loweraccumulation of radiolabeled LDL compared to nonimmune-balloonedrabbits. These results indicate that active immunization against LBPprovides an effective means by which the accumulation of LDL in theinjured arterial wall can be modified.

Example 20 Screening Agents in Humans Which Inhibit Binding Between LDLand LBPs

[0206] Human studies are carried out according to standard FDA protocolsfor testing of new drugs for safety (Phase I), efficacy (Phase II), andefficacy compared to other treatments (Phase III). Subjects, who areenrolled into studies after giving informed consent, are between theages of 18 and 70. Women who are pregnant, or likely to become pregnant,or subjects with diseases other than primary atherosclerosis, such ascancer, liver disease, or diabetes, are excluded. Subjects selected forstudy in FDA Phase II and Phase III trials have atherosclerotic diseasepreviously documented by standard techniques, such as ultrasound and/orangiography, or are known to be at high risk of atherosclerosis byvirtue of having at least one first degree relative with documentedatherosclerosis. Subjects themselves have normal or abnormal plasmalipids. Initial testing includes 20-50 subjects on active drug and 20-50subjects, matched for age, sex, and atherosclerotic status, on placebo.The number of subjects is pre-determined by the number needed forstatistical significance. Endpoints for inhibitor efficacy includesultrasound measurements of carotid artery thickness in high risksubjects, as well as in subjects with known carotid or coronary disease;atherosclerotic events; atherosclerotic deaths; and all-cause deaths inall subjects. Non-invasive analysis (carotid artery thickness byultrasound) as per Stadler (Med. and Biol. 22:25-34 (1996)) are carriedout at 6- to 12-month intervals for 3 years. Atherosclerotic events anddeaths, as well as all-cause deaths are tabulated at 3 years.

[0207] Oral dosage of drug in FDA Phase I trials ranges from 0.01 to 10gm/day, and is determined by results of animal studies, extrapolated ona per kg basis. Based on data obtained from Phase I studies, the doserange and frequency are narrowed in Phase II and III trials. Ifparenteral administration of drug is determined by animal studies to bethe only effective method, parenteral administration in human subjectsis tested by injection, as well as by the transdermal and nasalinsufflation routes. Testing of parenteral drug follows the same outlineas that for oral administration.

[0208] The optimal treatment schedule and dosage for humans is thusestablished.

Example 21 Treating an Individual Having Atherosclerosis with BHF-1

[0209] This example illustrates a method for treating an individualhaving atherosclerosis with an LBP fragment, e.g., BHF-1, so as todecrease the levels of arterially bound LDL in the individual. BHF-1 isobtained as described herein. The BHF-1 is administered to the mammalintravenously as a bolus or as an injection at a concentration of 0.5-10mg/kg body weight. Such administrations are repeated indefinitely inorder to prevent the development or progression of symptomaticatherosclerosis, just as is done currently with cholesterol-loweringdrugs. Stable subjects are examined twice yearly to evaluate the extentof any atherosclerotic disease by physical exam and non-invasivestudies, such as carotid artery thickness, ultrasound, and/or gammacamera imaging of the major arteries, to determine if atheroscleroticlesions are present, and, if previously present, have regressed orprogressed. Such a regimen results in treatment of the atherosclerosis.

[0210] Those skilled in the art will be able to ascertain using no morethan routine experimentation, many equivalents of the specificembodiments of the invention described herein. These and all otherequivalents are intended to be encompassed by the following claims.

What is claimed is:
 1. An isolated polynucleotide comprising a memberselected from the group consisting of: (a) a polynucleotide encoding thepolypeptide comprising the amino acid sequence as set forth in SEQ IDNO:1; (b) a polynucleotide encoding the polypeptide comprising the aminoacid sequence as set forth in SEQ ID NO:2; (c) a polynucleotide encodingthe polypeptide comprising the amino acid sequence as set forth in SEQID NO:3; (d) a polynucleotide encoding the polypeptide comprising theamino acid sequence as set forth in SEQ ID NO:4; (e) a polynucleotideencoding the polypeptide comprising the amino acid sequence as set forthin SEQ ID NO:5; (f) a polynucleotide encoding the polypeptide comprisingthe amino acid sequence as set forth in SEQ ID NO:6; (g) apolynucleotide encoding the polypeptide comprising the amino acidsequence as set forth in SEQ ID NO:7; (h) a polynucleotide encoding thepolypeptide comprising the amino acid sequence as set forth in SEQ IDNO:8; (i) a polynucleotide encoding the polypeptide comprising the aminoacid sequence as set forth in SEQ ID NO:9; (j) a polynucleotide capableof hybridizing to and which is at least about 95% identical to thepolynucleotide of (a)-(h) or (i) wherein the encoded polypeptide iscapable of binding to LDL; and (k) a biologically active fragment ofpolynucleotide (a)-(i) or (j) wherein the encoded polypeptide is capableof binding to LDL.
 2. An isolated polynucleotide of claim 1 wherein saidmember is selected from the group consisting of: (a) a polynucleotideencoding the polypeptide comprising the amino acid residues 8-22 (SEQ IDNO:19), 8-33 (SEQ ID NO:20), 23-33 (SEQ ID NO:21) or 208-217 (SEQ IDNO:22) of the amino acid sequence as set forth in SEQ ID NO:7; (b) apolynucleotide encoding the polypeptide comprising the amino acidresidues 14-43 (SEQ ID NO:23) or 38-43 (SEQ ID NO:24) of the amino acidsequence as set forth in SEQ ID NO:1 and SEQ ID NO:6; (c) apolynucleotide encoding the polypeptide comprising the amino acidresidues 105-120 (SEQ ID NO:25), 105-132 (SEQ ID NO:26), 121-132 (SEQ IDNO:27) or 211-220 (SEQ ID NO:28) of the amino acid sequence as set forthin SEQ ID NO:2; (d) a polynucleotide encoding the polypeptide comprisingthe amino acid residues 96-110 (SEQ ID NO:29) of the amino acid sequenceas set forth in SEQ ID NO:5; (e) a polynucleotide encoding thepolypeptide comprising the amino acid residues 53-59 (SEQ ID NO:41) ofthe amino acid sequence as set forth in SEQ ID NO:8; (f) apolynucleotide capable of hybridizing to and which is at least about 95%identical to the polynucleotide of (a)-(d) or (e) wherein the encodedpolypeptide is capable of binding to LDL; and (g) a biologically activefragment of polynucleotide (a)-(e) or (f) wherein the encodedpolypeptide is capable of binding to LDL.
 3. The polynucleotide of claim1 wherein said polynucleotide comprises the nucleic acid as set forth inSEQ ID NO:10.
 4. The polynucleotide of claim 1 wherein saidpolynucleotide comprises the nucleic acid as set forth in SEQ ID NO:11.5. The polynucleotide of claim 1 wherein said polynucleotide comprisesthe nucleic acid as set forth in SEQ ID NO:12.
 6. The polynucleotide ofclaim 1 wherein said polynucleotide comprises the nucleic acid as setforth in SEQ ID NO:13.
 7. The polynucleotide of claim 1 wherein saidpolynucleotide comprises the nucleic acid as set forth in SEQ ID NO:14.8. The polynucleotide of claim 1 wherein said polynucleotide comprisesthe nucleic acid as set forth in SEQ ID NO:15.
 9. The polynucleotide ofclaim 1 wherein said polynucleotide comprises the nucleic acid as setforth in SEQ ID NO:16.
 10. The polynucleotide of claim 1 wherein saidpolynucleotide comprises the nucleic acid as set forth in SEQ ID NO:17.11. The polynucleotide of claim 1 wherein said polynucleotide comprisesthe nucleic acid as set forth in SEQ ID NO:18.
 12. The polynucleotide ofclaim 2 wherein said polynucleotide comprises the nucleic acid as setforth in SEQ ID NO:30.
 13. The polynucleotide of claim 2 wherein saidpolynucleotide comprises the nucleic acid as set forth in SEQ ID NO:31.14. The polynucleotide of claim 2 wherein said polynucleotide comprisesthe nucleic acid as set forth in SEQ ID NO:32.
 15. The polynucleotide ofclaim 2 wherein said polynucleotide comprises the nucleic acid as setforth in SEQ ID NO:33.
 16. The polynucleotide of claim 2 wherein saidpolynucleotide comprises the nucleic acid as set forth in SEQ ID NO:34.17. The polynucleotide of claim 2 wherein said polynucleotide comprisesthe nucleic acid as set forth in SEQ ID NO:35.
 18. The polynucleotide ofclaim 2 wherein said polynucleotide comprises the nucleic acid as setforth in SEQ ID NO:36.
 19. The polynucleotide of claim 2 wherein saidpolynucleotide comprises the nucleic acid as set forth in SEQ ID NO:37.20. The polynucleotide of claim 2 wherein said polynucleotide comprisesthe nucleic acid as set forth in SEQ ID NO:38.
 21. The polynucleotide ofclaim 2 wherein said polynucleotide comprises the nucleic acid as setforth in SEQ ID NO:39.
 22. The polynucleotide of claim 2 wherein saidpolynucleotide comprises the nucleic acid as set forth in SEQ ID NO:40.23. The polypeptide of claim 2 wherein said polynucleotide comprises thenucleic acid as set forth in SEQ ID NO:42.
 24. The polynucleotide ofclaim 1 wherein said polynucleotide is selected from the groupconsisting of DNA and RNA.
 25. The polynucleotide of claim 1 whereinsaid polynucleotide is genomic DNA.
 26. A recombinant vector comprisingsaid polynucleotide of claim
 1. 27. A cell comprising said recombinantvector of claim
 26. 28. A method for producing an LDL binding proteincomprising culturing a cell of claim 27 under conditions that permitexpression of said LDL binding protein.
 29. An isolated polypeptidecomprising a member selected from the group consisting of: (a) apolypeptide having the amino acid sequence as set forth in SEQ ID NO:1;(b) a polypeptide having the amino acid sequence as set forth in SEQ IDNO:2; (c) a polypeptide having the amino acid sequence as set forth inSEQ ID NO:3; (d) a polypeptide having the amino acid sequence as setforth in SEQ ID NO:4; (e) a polypeptide having the amino acid sequenceas set forth in SEQ ID NO:5; (f) a polypeptide having the amino acidsequence as set forth in SEQ ID NO:6; (g) a polypeptide having the aminoacid sequence as set forth in SEQ ID NO:7; (h) a polypeptide having theamino acid sequence as set forth in SEQ ID NO:8; (i) a polypeptidehaving the amino acid sequence as set forth in SEQ ID NO:9; (j) apolypeptide which is at least about 95% identical to the polypeptide of(a)-(h) or (i) wherein said polypeptide is capable of binding to LDL;and (k) a biologically active fragment of polypeptide (a)-(i) or (j)wherein said fragment is capable of binding to LDL.
 30. An isolatedpolypeptide of claim 29 wherein said member is selected from the groupconsisting of: (a) a polypeptide having the amino acid residues 8-22(SEQ ID NO:19), 8-33 (SEQ ID NO:20), 23-33 (SEQ ID NO:21) or 208-217(SEQ ID NO:22) of the amino acid sequence as set forth in SEQ ID NO:7;(b) a polypeptide having the amino acid residues 14-43 (SEQ ID NO:23) or38-43 (SEQ ID NO:24) of the amino acid sequence as set forth in SEQ IDNO:1 and SEQ ID NO:6; (c) a polypeptide having the amino acid residues105-120 (SEQ ID NO:25), 105-132 (SEQ ID NO:26), 121-132 (SEQ ID NO:27)or 211-220 (SEQ ID NO:28) of the amino acid sequence as set forth in SEQID NO:2; (d) a polypeptide having the amino acid residues 96-110 (SEQ IDNO:29) of the amino acid sequence as set forth in SEQ ID NO:5; (e) apolypeptide having the amino acid residues 53-59 (SSEQ ID NO:41) of theamino acid sequence as set forth in SEQ ID NO:8; (f) a polypeptide whichis at least about 95% identical to the polypeptide of (a)-(d) or (e)wherein said polypeptide is capable of binding to LDL; and (g) abiologically active fragment of polypeptide (a)-(e) or (f) wherein saidfragment is capable of binding to LDL.
 31. A method for determining ifan animal is at risk for atherosclerosis, comprising: providing ananimal; and evaluating an aspect of LBP metabolism or structure in saidanimal, an abnormality in said aspect of LBP metabolism or structurebeing diagnostic of being at risk for atherosclerosis.
 32. The method ofclaim 31 wherein said LBP is selected from the group consisting ofLBP-1, LBP-2 and LBP-3.
 33. The method of claim 31 wherein said aspectof LBP metabolism is the ability of said LBP to bind to LDL.
 34. Themethod of claim 31 wherein said aspect of LBP metabolism is the abilityof said LBP to bind to an arterial extracellular matrix structuralcomponent.
 35. The method of claim 34 wherein said component is selectedfrom the group consisting of proteoglycans, elastin, collagen,fibronectin, vitronectin and integrins.
 36. The method of claim 31wherein said risk is a reduced risk as compared to a normal animal. 37.The method of claim 36 wherein said abnormality results in an inactiveLBP polypeptide.
 38. The method of claim 31 wherein said risk is anincreased risk as compared to a normal animal.
 39. The method of claim38 wherein said abnormality results in an LBP polypeptide that hashigher activity than native LBP polypeptide.
 40. The method of claim 31wherein said animal is a prenatal animal.
 41. A method for evaluating anagent for use in treating atherosclerosis, comprising: providing a testcell, cell-free system or animal; providing an agent; administering saidagent to said test cell, cell-free system or animal in a therapeuticallyeffective amount; and evaluating the effect of said agent on an aspectof LBP metabolism or structure, a change in said aspect of LBPmetabolism or structure being indicative of the usefulness of said agentin treating atherosclerosis.
 42. The method of claim 41 wherein saidtest cell, cell-free system or animal has a wild type pattern of LBPmetabolism.
 43. The method of claim 41 wherein said test cell, cell-freesystem or animal has a non-wild type pattern of LBP metabolism.
 44. Themethod of claim 41 wherein said LBP is selected from the groupconsisting of LBP-1, LBP-2 and LBP-3.
 45. The method of claim 41 whereinsaid agent comprises LBP-1, LBP-2 or LBP-3 polypeptide or a biologicallyactive fragment or analog thereof.
 46. The method of claim 41 whereinsaid agent is selected from the group consisting of a polypeptidecomprising an amino acid sequence as set forth in FIGS. 1-8 and 9 (SEQID NOS:1-9).
 47. The method of claim 41 wherein said agent comprises anucleic acid encoding LBP-1, LBP-2 or LBP-3 polypeptide or abiologically active fragment or analog thereof.
 48. The method of claim41 wherein said agent is selected from the group consisting of a nucleicacid comprising a nucleotide sequence as set forth in FIGS. 10-17 and 18(SEQ ID NOS:10-18).
 49. The method of claim 41 wherein said agentcomprises a nucleic acid encoding an LBP regulatory sequence or abiologically active fragment thereof.
 50. The method of claim 41 whereinsaid agent is selected from the group consisting of a binding moleculefor said LBP polypeptide and a binding molecule for said LBP nucleicacid.
 51. The method of claim 41 wherein said agent is an antisensenucleic acid or analog thereof.
 52. The method of claim 41 wherein saidagent is selected from the group consisting of a mimetic of said LBP anda mimetic of a binding molecule of said LBP.
 53. The method of claim 41wherein said agent is a polyclonal or monoclonal antibody, or fragmentthereof, that can immunoreact with an LBP polypeptide.
 54. The method ofclaim 41 wherein said agent is selected from the group consisting of anatural antibody, a recombinant antibody, a chimeric antibody and ahumanized antibody that can immunoreact with an LBP polypeptide.
 55. Themethod of claim 41 wherein said agent is a natural ligand for said LBP.56. The method of claim 41 wherein said agent is an artificial ligandfor said LBP.
 57. The method of claim 41 wherein said agent is selectedfrom the group consisting of an antagonist, an agonist and a superagonist.
 58. The method of claim 41 wherein said agent is administeredto a member selected from the group consisting of a transgenic cell anda transgenic animal.
 59. The method of claim 41 wherein said agent isadministered to said test cell or cell-free system in vitro, and if saidchange in said aspect of said LBP metabolism occurs, then furtheradministering said agent to a test animal in a therapeutically effectiveamount and evaluating the in vivo effect of said agent on an aspect ofLBP metabolism.
 60. The agent identified in claim
 41. 61. A method forevaluating an agent for the ability to alter the binding of LBPpolypeptide to a binding molecule, comprising: providing an agent;providing LBP polypeptide; providing a binding molecule; combining saidagent, said LBP polypeptide and said binding molecule; and detecting theformation of a complex comprising said LBP polypeptide and said bindingmolecule, an alteration in the formation of said complex in the presenceof said agent as compared to in the absence of said agent beingindicative of said agent altering the binding of said LBP polypeptide tosaid binding molecule.
 62. The method of claim 61 wherein said LBPpolypeptide is selected from the group consisting of LBP-1, LBP-2 andLBP-3 polypeptide.
 63. The method of claim 61 wherein the altering ofthe binding of said LBP polypeptide to said binding molecule isinhibiting the binding.
 64. The method of claim 61 wherein the alteringof the binding of said LBP polypeptide to said binding molecule ispromoting the binding.
 65. The method of claim 61 wherein said bindingmolecule is selected from the group consisting of native LDL andmodified LDL.
 66. The method of claim 61 wherein said binding moleculeis an arterial extracellular matrix structural component.
 67. The agentidentified in claim
 61. 68. A method for evaluating an agent for theability to bind to an LBP polypeptide, comprising: providing an agent;providing an LBP polypeptide; contacting said agent with said LBPpolypeptide; and evaluating the ability of said agent to bind to saidLBP polypeptide.
 69. The method of claim 68 wherein said LBP polypeptideis selected from the group consisting of LBP-1, LBP-2 and LBP-3polypeptide.
 70. The agent identified in claim
 68. 71. A method forevaluating an agent for the ability to bind to a nucleic acid encodingan LBP regulatory sequence, comprising: providing an agent; providing anucleic acid encoding an LBP regulatory sequence; contacting said agentwith said nucleic acid; and evaluating the ability of said agent to bindto said nucleic acid.
 72. The method of claim 71 wherein said LBPregulatory sequence is selected from the group consisting of LBP-1,LBP-2 and LBP-3.
 73. The agent identified in claim
 71. 74. A method fortreating atherosclerosis in an animal, comprising: providing an animalin need of treatment for atherosclerosis; providing an agent capable ofaltering an aspect of LBP structure or metabolism; administering saidagent to said animal in a therapeutically effective amount such thattreatment of said atherosclerosis occurs.
 75. The method of claim 74wherein said agent is an LBP polypeptide.
 76. The method of claim 75wherein said LBP polypeptide is LBP-1, LBP-2 or LBP-3 polypeptide or abiologically active fragment or analog thereof.
 77. The method of claim76 wherein said agent is selected from the group consisting of apolypeptide comprising an amino acid sequence as set forth in SEQ IDNOS:1-8 and
 9. 78. The method of claim 76 wherein said agent is selectedfrom the group consisting of a polypeptide comprising amino acidresidues 8-22 (SEQ ID NO:19), 8-33 (SEQ ID NO:20), 23-33 (SEQ ID NO:21)or 208-217 (SEQ ID NO:22) of human LBP-2 as depicted in SEQ ID NO:7;amino acid residues 14-43 (SEQ ID NO:23) or 38-43 (SEQ ID NO:24) ofrabbit or human LBP-1 as depicted in SEQ ID NO:1 and SEQ ID NO:6; aminoacid residues 105-120 (SEQ ID NO:25), 105-132 (SEQ ID NO:26), 121-132(SEQ ID NO:27) or 211-220 (SEQ ID NO:28) of rabbit LBP-2 as depicted inSEQ ID NO:2; amino acid residues 96-110 (SEQ ID NO:29) of rabbit LBP-3as depicted in SEQ ID NO:5; and amino acid residues 53-59 (SEQ ID NO:41)as set forth in SEQ ID NO:8.
 79. The method of claim 74 wherein saidagent is a polypeptide of no more than about 50 amino acid residues inlength.
 80. The method of claim 74 wherein said agent is a polypeptidehaving an amino acid sequence that includes at least about 20% acidicamino acid residues.
 81. The method of claim 74 wherein said agent isselected from the group consisting of a homopolymer of an acidic aminoacid or analog thereof, and a heteropolymer of one or more acidic aminoacids and one or more other amino acids or analogs thereof.
 82. Themethod of claim 74 wherein said agent is selected from the groupconsisting of poly(glu), poly(asp) and poly(glu asp).
 83. The method ofclaim 74 wherein said agent is selected from the group consisting ofpoly(glu N), poly(asp N) and poly(glu asp N).
 84. The method of claim 74wherein said agent is poly(glu) of no more than about 10 amino acidresidues in length.
 85. The method of claim 74 wherein said agent is anLBP nucleic acid or a biologically active fragment or analog thereof.86. The method of claim 85 wherein said LBP nucleic acid comprises anucleic acid encoding LBP-1, LBP-2 or LBP-3 polypeptide or abiologically active fragment or analog thereof.
 87. The method of claim86 wherein said agent is selected from the group consisting of a nucleicacid comprising a nucleotide sequence as set forth in SEQ ID NOS:10-17and
 18. 88. The method of claim 74 wherein said agent is an antisensenucleic acid or analog thereof.
 89. A method for treating an animal atrisk for atherosclerosis, comprising: providing an animal at risk foratherosclerosis; providing an agent capable of altering an aspect of LBPstructure or metabolism; and administering said agent to said animal ina therapeutically effective amount such that treatment of said animaloccurs.
 90. A method for treating a cell having an abnormality instructure or metabolism of LBP, comprising: providing a cell having anabnormality in structure or metabolism of LBP; providing an agentcapable of altering an aspect of LBP structure or metabolism; andadministering said agent to said cell in a therapeutically effectiveamount such that treatment of said cell occurs.
 91. The method of claim90 wherein said LBP is selected from the group consisting of LBP-1,LBP-2 and LBP-3.
 92. The method of claim 90 wherein said cell isobtained from a cell culture or tissue culture.
 93. The method of claim90 wherein said cell is obtained from an embryo fibroblast.
 94. Themethod of claim 90 wherein said cell is part of an animal.
 95. Themethod of claim 94 wherein said animal is a non-human transgenic animal.96. A pharmaceutical composition for treating atherosclerosis in ananimal, comprising: a therapeutically effective amount of an agent, saidagent being capable of altering an aspect of LBP metabolism or structurein said animal so as to result in treatment of said atherosclerosis; anda pharmaceutically acceptable carrier.
 97. The pharmaceuticalcomposition of claim 96 wherein said agent is an LBP polypeptide ornucleic acid, or biologically active fragment or analog thereof.
 98. Thepharmaceutical composition of claim 96 wherein said agent is apolypeptide of no more than about 50 amino acid residues in length. 99.The pharmaceutical composition of claim 96 wherein said agent is apolypeptide having an amino acid sequence that includes at least about20% acidic amino acid residues.
 100. A vaccine composition for treatingatherosclerosis in an animal, comprising: a therapeutically effectiveamount of an agent, said agent being capable of altering an aspect ofLBP metabolism or structure in said animal so as to result in treatmentof said atherosclerosis; and a pharmaceutically acceptable carrier. 101.A method for diagnosing atherosclerotic lesions in an animal,comprising: providing an animal; providing a labeled agent capable ofbinding to LBP present in atherosclerotic lesions; administering saidlabeled agent to said animal under conditions which allow said labeledagent to interact with said LBP so as to result in labeled LBP; anddetermining the localization or quantification of said labeled LBP byimaging so as to diagnose the presence of atherosclerotic lesions insaid animal.
 102. The method of claim 101 wherein said LBP is selectedfrom the group consisting of LBP-1, LBP-2 and LBP-3.
 103. The method ofclaim 101 wherein said imaging is selected from the group consisting ofmagnetic resonance imaging, gamma camera imaging, single photon emissioncomputed tomographic (SPECT) imaging and positron emission tomography(PET).
 104. A method for immunizing an animal against an LBP or fragmentor analog thereof, comprising: providing an animal having LDL; providingan LBP or fragment or analog thereof; administering said LBP or fragmentor analog thereof to said animal so as to stimulate antibody productionby said animal to said LBP or fragment or analog thereof such thatbinding of said LBP to said LDL is altered.
 105. The method of claim 104wherein binding of said LBP to said LDL is decreased.
 106. A method ofmaking a fragment or analog of LBP polypeptide, said fragment or analoghaving the ability to bind to modified LDL and native LDL, comprising:providing an LBP polypeptide; altering the sequence of said LBPpolypeptide; and testing said altered LBP polypeptide for the ability tobind to modified LDL and native LDL.
 107. The method of claim 106wherein said LBP is selected from the group consisting of LBP-1, LBP-2and LBP-3.
 108. The method of claim 106 wherein said altered LBPpolypeptide is selected from the group consisting of an antagonist, anagonist and a super agonist.
 109. A method for isolating a cDNA encodingan LBP, comprising: providing a cDNA library; screening said cDNAlibrary for a cDNA encoding a polypeptide which binds to native LDL andmodified LDL; and isolating said cDNA which encodes said polypeptide,said cDNA encoding an LBP.